A method for producing butylenediamine by double cell coupling fermentation, and a strain

Abstract

The present application belongs to the field of biotechnology and synthetic biology, and particularly relates to a method and strains for producing butanediamine by double-cell coupling fermentation. The present application constructs an arginine-producing strain that can only utilize glucose in the system, an arginine-producing strain that has metabolic interference and cannot utilize xylose, and a butanediamine-converting strain that completely loses the ability to utilize glucose and can only utilize xylose, thereby fundamentally avoiding carbon metabolic repression and reducing the metabolic burden of a single strain. Meanwhile, by using a mixed fermentation system combined with fermentation process regulation, the problems of high-concentration feedback inhibition of intermediate metabolic product arginine and cell toxicity of end product butanediamine are effectively solved, and the synergistic utilization of glucose and xylose in lignocellulose hydrolysate can be realized. The butanediamine yield in a 5 L fermenter can reach more than 45.2 g / L, and the sugar conversion rate is 0.22 g / g. The present application provides a new strain and technical path for green manufacturing of bio-based butanediamine, and has a broad industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and synthetic biology, and specifically relates to a method and strain for producing butanediamine using two-cell coupled fermentation. Background Technology

[0002] 1,4-Butanediamine (Putrescine, also known as putrescine) is an important C4 platform compound, primarily used in the synthesis of high-performance polyamide nylon 46. Nylon 46, due to its high heat resistance (melting point 295℃) and excellent wear and impact resistance, is widely used in high-end manufacturing fields such as electronic components, automotive engine parts, and precision bearings. With the rapid development of new energy vehicles and the electronic information industry, the market demand and strategic value of 1,4-butanediamine continue to increase.

[0003] Traditional butanediamine production employs a petroleum-based chemical synthesis route using acrylonitrile and hydrogen cyanide. This route utilizes highly toxic hydrogen cyanide as a raw material, requires stringent reaction conditions and significant equipment investment, and generates substantial carbon emissions, contradicting the principles of green and sustainable development. Utilizing microbial cell factories to convert renewable biomass resources into butanediamine has become the mainstream development direction to replace traditional chemical methods.

[0004] Lignocellulose is the most abundant renewable carbon source, with my country producing approximately 900 million tons of crop straw annually. Hydrolysis of its cellulose and hemicellulose yields a mixed fermentable sugar consisting of 60-70% glucose and 20-30% xylose. This sugar can be efficiently used in the production of butanediamine, solving the problem of agricultural waste pollution and providing a low-cost raw material for the bio-based materials industry.

[0005] However, biofermentation using lignocellulose hydrolysate faces two major technical bottlenecks: Bottleneck 1, the repression effect of carbon decomposition metabolites. Industrial microorganisms such as *E. coli* preferentially utilize glucose in a mixed sugar environment. Glucose inhibits the transcription and transport system activity of genes related to xylose utilization. The molecular mechanism is that after glucose enters the cell via the phosphotransferase system, it reduces intracellular cAMP levels and cAMP receptor protein activity, thereby inhibiting xylose operon transcription. This results in xylose being slowly consumed only after glucose is depleted, prolonging the fermentation cycle and reducing raw material utilization. Bottleneck 2, excessive metabolic burden on a single strain. If a single engineered strain completes the entire conversion process from mixed sugars to butanediamine, the strain must simultaneously overcome carbon metabolism repression, simultaneously utilize glucose and xylose, synthesize L-arginine from glucose through multiple enzymatic reactions, catalyze the two-step conversion of arginine to butanediamine, and tolerate feedback inhibition from products and intermediates. This excessive metabolic load leads to slow strain growth, poor plasmid stability, and low product yield.

[0006] Existing technologies often alleviate carbon metabolism repression by knocking out genes such as ptsG and glk, or improve xylA / xylB utilization efficiency by overexpressing xylA / xylB, or use whole-cell catalysis to avoid sugar source utilization problems. However, none of these technologies can simultaneously solve the core problems of efficient utilization of whole sugar and optimization of metabolic division of labor. Summary of the Invention

[0007] The technical problem to be solved by this invention is to address three common industry problems in the fermentation process of lignocellulose mixed sugars: inhibition of carbon decomposition metabolites, feedback inhibition of arginine intermediates, and cytotoxicity of 1,4-butanediamine final products. This invention provides a method for producing 1,4-butanediamine using two-cell coupled fermentation, constructing an efficient and stable two-cell fermentation system to achieve efficient conversion of lignocellulose hydrolysate to 1,4-butanediamine.

[0008] The technical problem that this invention also aims to solve is to provide an arginine-producing strain and a butylene-converting strain for the production of butanediamine through two-cell coupled fermentation.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] A method for producing butanediamine using two-cell coupled fermentation includes the following steps:

[0011] (1) First stage: The seed culture of arginine-producing strain A was inoculated into a mixed carbon source medium containing glucose and xylose and fermented until the arginine concentration in the fermentation broth reached 30~35 g / L. During the fermentation process, glucose was added to maintain the glucose concentration at 5~15 g / L.

[0012] (2) Second stage: According to the cell biomass ratio of arginine production strain A to butanediamine conversion strain B = 5:1~8:1, continue to inoculate the seed liquid of butanediamine conversion strain B into the fermentation broth of step (1), and stop the flow of glucose, and instead flow xylose to maintain the xylose concentration at 5~10 g / L, and continue fermentation to convert arginine into butanediamine.

[0013] The above method is based on two core design principles: (a) substrate-level carbon source specialization—the two strains independently utilize glucose and xylose, completely eliminating the carbon repression effect (CCR) at the substrate inlet; (b) product-level metabolic specialization—arginine synthesis and butanediamine conversion are completed by the two strains, simultaneously relieving arginine feedback inhibition and butanediamine cytotoxicity. Through the coupling of these two levels of specialization, this method simultaneously solves the three major common technical bottlenecks that have long existed in the fermentation of mixed sugars from lignocellulose.

[0014] The arginine-producing strain A was obtained by using Escherichia coli BL21(DE3) as the starting strain, knocking out arginine degradation-related genes speA, adiA, and astA, knocking out the global negative regulator argR, integrating the argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, pntAB, and lysE genes, and replacing the natural promoter of the sucA gene with the PrpsT promoter.

[0015] The nucleotide sequences of the arginine degradation-related genes speA, adiA, and astA are shown in SEQ ID NO. 12-14, the nucleotide sequence of the global negative regulator argR is shown in SEQ ID NO. 18, and the nucleotide sequences of the argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, pntAB, and lysE genes are shown in SEQ ID NO. 20, SEQ ID NO. 25-28, SEQ ID NO. 32-33, SEQ ID NO. 42, and SEQ ID NO. 50, respectively; the nucleotide sequence of the PrpsT promoter is shown in SEQ ID NO. 46.

[0016] The butanediamine-transformed strain B was obtained by knocking out the ptsG, glk, and manZ genes and overexpressing the arginase gene ARG and the ornithine decarboxylase gene speC, using Escherichia coli BL21(DE3) as the starting strain.

[0017] Specifically, the ptsG gene encodes the IICB-Glc component of the glucose-specific PTS transport system; its deletion blocks the primary pathway for glucose to enter cells via the PTS system. The glk gene encodes glucokinase, preventing the phosphorylation of trace amounts of glucose entering the cell via other pathways and thus preventing them from entering the glycolysis pathway. The manZ gene encodes the IID component of the mannose PTS system; knocking out this gene eliminates a secondary pathway for glucose uptake (the ManXYZ system). After the combined knockout of the ptsG, glk, and manZ genes, the strain completely loses its ability to uptake and metabolize exogenous glucose while retaining the complete xylose metabolic pathway, allowing it to exclusively utilize xylose for growth and reproduction in a mixed glucose-xylose medium. This strain can form a completely independent carbon source utilization division of labor with glucose-utilizing strains, fundamentally avoiding the repression effect of carbon decomposition metabolites in mixed sugar systems. Simultaneously, through the metabolic functional separation from arginine-synthetic strains, it provides the strain prerequisites for the spatiotemporal separation of arginine synthesis and butanediamine conversion, the relief of arginine feedback inhibition, and the reduction of butanediamine cytotoxicity.

[0018] To further enhance xylose utilization rate in the strain, the xylose isomerase encoding gene *xylA* and the xylose kinase encoding gene *xylB* can be overexpressed in the aforementioned three-gene knockout strain. These two genes can be cloned into expression plasmids such as *pET28a-T7-xylAB*, driven by constitutive promoters, or integrated into specific sites in the genome for stable expression. Overexpression of the *xylAB* gene can enhance xylose utilization efficiency, improve cell growth and arginine conversion, ensure immediate and efficient conversion of extracellular arginine, and further enhance the relief of arginine feedback inhibition; simultaneously, it can improve the synthesis and efflux efficiency of butanediamine, reducing cytotoxicity caused by intracellular product accumulation.

[0019] The nucleotide sequences of the ptsG, glk, and manZ genes are shown in SEQ ID NO.1-3, and the nucleotide sequences of the arginase ARG and ornithine decarboxylase speC are shown in SEQ ID NO.10-11.

[0020] The nucleotide sequences of the xylose isomerase encoding gene xylA and the xylose kinase encoding gene xylB are shown in SEQ ID NO. 7~8, respectively.

[0021] Specifically, the argJ gene is integrated into the lacI site driven by the Pprc promoter; the argC, argB, argD, and argF genes are fused sequentially and then integrated into the yghX pseudogene site driven by the Pprc promoter; the argG and argH genes are fused sequentially and then integrated into the ygaY pseudogene site driven by the Pprc promoter; the pyrAA and pyrAB genes are fused sequentially and then integrated into the yciV pseudogene site driven by the Pprc promoter; the pntAB gene is integrated into the yjiP pseudogene site driven by the Pprc promoter; and the lysE gene is integrated into the ilvG site driven by the Pprc promoter.

[0022] The nucleotide sequence of the Pprc promoter is shown in SEQ ID NO.21.

[0023] In step (2), 0.5~0.8 mM IPTG is added to the fermentation broth from step (1), and the temperature of the second stage is controlled at 33℃~37℃ and the dissolved oxygen at 30%~50%.

[0024] The first stage of fermentation focuses on arginine accumulation: Fermentation medium is added to the fermenter, preferably a hydrolysate of lignocellulose from corn straw, rice straw, or wheat straw after activated carbon detoxification; a mixed fermentation medium of glucose and xylose can also be used. Seed culture of strain A is inoculated at a rate of 5-10% v / v, and the fermentation temperature is controlled at 37℃, pH 6.8-7.2, and dissolved oxygen at 30%-40%. When the initial glucose concentration drops to 5-10 g / L, a glucose feed solution is added to maintain a glucose concentration of 5-15 g / L. Fermentation continues for 15-25 hours until the arginine concentration reaches 30-35 g / L. In this stage, only strain A is inoculated, allowing it to exclusively utilize glucose in the system to synthesize arginine, without metabolic interference from xylose utilization or the toxic effects of butanediamine end products. This allows for the rapid accumulation of high concentrations of arginine precursors. Simultaneously, through extracellular secretion design, the intracellular feedback inhibition of arginine is initially alleviated, ensuring the continuous and efficient operation of the synthetic pathway.

[0025] The second stage of fermentation is a dual-strain coupled fermentation: after the arginine concentration reaches the target, the fermentation temperature is adjusted to 33-37℃ (preferably 35℃) and dissolved oxygen to 30%-50%. Strawberry B seed culture is inoculated at a cell biomass ratio of strain A:strain B = 5:1 to 8:1 (preferably 5:1), and IPTG is added at a final concentration of 0.5-0.8 mM (preferably 0.5 mM) to induce ARG and speC gene expression. Adapting to the sugar component utilization logic of the lignocellulose hydrolysate, glucose feeding is stopped, and xylose feeding solution is switched to maintain a xylose concentration of 5-10 g / L in the system, and fermentation continues for 48 h. After inoculation with strain B in this stage, strain A continuously utilizes glucose to synthesize arginine, while strain B specifically utilizes xylose for growth and expresses invertase systems. The utilization of the two carbon sources is completely independent and does not interfere with each other, fundamentally avoiding the repression effect of carbon decomposition metabolites. Strain B can promptly and efficiently convert arginine in the system into butanediamine, maintaining a consistently low concentration of arginine in the fermentation system. This continuously relieves the feedback inhibition of strain A by high-concentration arginine, achieving continuous synthesis and targeted conversion of arginine. Simultaneously, the synthesis of butanediamine is entirely completed by strain B, and the arginine synthesis metabolism of strain A is never affected by the feedback inhibition or toxicity of the final product butanediamine, thus stably maintaining the precursor supply capacity. Furthermore, the staged temperature and dissolved oxygen regulation can adapt to the optimal growth and conversion conditions of strain B, enhancing its tolerance to butanediamine and further reducing the impact of product cytotoxicity on overall fermentation efficiency.

[0026] In the above-described method for producing butanediamine using two-cell coupled fermentation, the multi-level feedback inhibition relief and toxicity detoxification strategies form a closed-loop synergistic effect: the higher the arginine efflux efficiency of strain A, the more timely the arginine is transferred to the extracellular space, the more abundant the substrate available to strain B, and the higher the catalytic conversion efficiency of strain B; the higher the catalytic conversion efficiency of strain B, the lower the extracellular arginine concentration, the stronger the driving force for arginine efflux of strain A, the more thorough the feedback inhibition relief, and the higher the arginine yield. Simultaneously, the efficient synthesis and rapid efflux of butanediamine in strain B gradually increases the butanediamine concentration in the fermentation broth. However, strain A, because its metabolic function does not involve butanediamine synthesis, is not directly affected by butanediamine accumulation in its arginine synthesis pathway; strain B maintains a certain level of activity through enzyme source optimization, catalytic rate matching, efflux enhancement, and adjustment of low-temperature, low-dissolved-oxygen process parameters.

[0027] This two-phase distribution of "low arginine concentration maintenance - high butanediamine accumulation," and the positive metabolic coupling between "precursor supply - final product synthesis," cannot be achieved in single-strain fermentation systems or simple superimposed two-strain systems. Experimental data show that the yield of butanediamine by direct fermentation of a single strain is only 14.6~22.1 g / L, and the strain's growth is severely limited (OD). 600 (8.2 only); while in the two-cell coupling system of the present invention, strain A still maintains good growth status (OD) when the butanediamine yield reaches 40.9~45.2 g / L or higher. 600 =42.3), with a total sugar utilization rate exceeding 98%. The above data fully demonstrate the significant advantages of the dual-cell coupling system of the present invention in relieving feedback inhibition and cytotoxicity, representing a substantial breakthrough over the bottlenecks of existing technologies.

[0028] In a second aspect, the present invention provides an arginine-producing strain for the two-cell coupled fermentation production of butanediamine as described in the first aspect. The arginine-producing strain is obtained by using Escherichia coli BL21(DE3) as the starting strain, knocking out arginine degradation-related genes speA, adiA, and astA, knocking out the global negative regulator argR, integrating the argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, and lysE genes, and replacing the natural promoter of the sucA gene with the PrpsT promoter.

[0029] The nucleotide sequences of the arginine degradation-related genes speA, adiA, and astA are shown in SEQ ID NO. 12-14, the nucleotide sequence of the global negative regulator argR is shown in SEQ ID NO. 18, and the nucleotide sequences of the argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, pntAB, and lysE genes are shown in SEQ ID NO. 20, SEQ ID NO. 25-28, SEQ ID NO. 32-33, SEQ ID NO. 42, and SEQ ID NO. 50, respectively; the nucleotide sequence of the PrpsT promoter is shown in SEQ ID NO. 46.

[0030] Specifically, arginine-producing strain A is a metabolically engineered *Escherichia coli* BL21(DE3), or *Corynebacterium glutamicum* can be used. It can efficiently synthesize and secrete L-arginine using glucose as a single carbon source. Its construction strategy includes:

[0031] (i) Degradation blocking pathway: Degradation pathways were blocked by knocking out arginine degradation-related genes speA, adiA and astA.

[0032] (ii) Relief of feedback inhibition: knock out the global negative regulator argR, heterologously express the argJ gene from Corynebacterium glutamicum and replace the function of argE, thereby relieving the allosteric inhibition of acetylglutamate synthase by argine and global transcriptional repression.

[0033] (iii) Enhancement of the synthesis pathway: overexpression of argB, argC, argD, argF, argG, and argH genes in the argine synthesis pathway, and overexpression of pyrAA / pyrAB and pntAB genes to increase NADPH supply.

[0034] (iv) Dynamic regulation of TCA carbon flux: Growth-dependent promoters were used to regulate sucA expression to optimize the allocation of TCA cycle carbon flux, enabling autonomous switching of carbon flux at the α-ketoglutarate node from cell growth to arginine synthesis.

[0035] (v) Enhancement of the efflux system: Overexpression of the arginine efflux transporter gene lysE (from Corynebacterium glutamicum) rapidly pumps intracellular arginine out, reducing the negative impact of product accumulation on strain growth.

[0036] The core advantage of arginine-producing strain A lies in its exclusive use of glucose for arginine synthesis, without bearing the metabolic burden of xylose utilization and butanediamine conversion. Through the synergistic effect of arginine transporter overexpression and feedback inhibition relief, the synthesized arginine can be rapidly secreted extracellularly, maintaining a stable arginine supply capacity throughout the entire fermentation cycle.

[0037] Thirdly, the present invention provides a butanediamine transforming strain for the two-cell coupled fermentation production of butanediamine as described in the first aspect. It is obtained by using Escherichia coli BL21(DE3) as the starting strain, by knocking out the ptsG, glk, and manZ genes, and overexpressing the arginase gene ARG and the ornithine decarboxylase gene speC.

[0038] The nucleotide sequences of the ptsG, glk, and manZ genes are shown in SEQ ID NO. 1-3, and the nucleotide sequences of the arginase ARG and ornithine decarboxylase speC are shown in SEQ ID NO. 10-11.

[0039] The butanediamine-transformed strain B also overexpresses the xylose isomerase encoding gene xylA and the xylose kinase encoding gene xylB.

[0040] The nucleotide sequences of the xylose isomerase encoding gene xylA and the xylose kinase encoding gene xylB are shown in SEQ ID NO. 7~8, respectively.

[0041] Butylene diamine transformant strain B is the xylose-specific recombinant Escherichia coli BL21-ΔptsGΔglkΔmanZ (or its xylAB enhanced strain) constructed in this invention, and its construction strategy includes:

[0042] (I) Complete blocking of glucose utilization: By knocking out the ptsG, glk, and manZ genes, the main pathway of the PTS system, the micro-glucose phosphorylation pathway, and the ManXYZ secondary uptake pathway are blocked, respectively, causing strain B to completely lose its ability to take up and metabolize glucose. At the same time, the complete xylose metabolic pathway is preserved, allowing it to exclusively utilize xylose for growth in a medium where glucose and xylose coexist, fundamentally avoiding the repression effect of carbon catabolites. Furthermore, overexpression of xylA and xylB can enhance the xylose utilization rate.

[0043] (2) Dual T7 independent promoter expression system: The dual T7 promoter co-expression plasmid pET28a-T7-ARG-T7-speC was constructed. ARG (preferably derived from bovine liver and codon optimized) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and SpeC catalyzes the decarboxylation of L-ornithine to generate 1,4-butanediamine and CO2. The two genes are regulated by independent T7 promoters. The expression ratio of ARG and SpeC can be flexibly adjusted by the IPTG concentration to achieve rate matching of each step of arginine uptake, hydrolysis, decarboxylation and efflux of 1,4-butanediamine, reduce the intracellular retention of intermediate and final products, and reduce cytotoxicity.

[0044] Beneficial effects:

[0045] (1) This invention proposes a method for producing butanediamine through two-cell coupled fermentation. The metabolic modules for glucose-to-arginine synthesis and arginine-to-butanediamine conversion are distributed to two dedicated engineered bacteria. This constructs an arginine-producing strain that exclusively utilizes glucose to synthesize arginine without xylose utilization metabolic interference, and a butanediamine-converting strain that completely loses its glucose utilization ability and exclusively utilizes xylose. This fundamentally avoids the carbon metabolism repression effect and reduces the metabolic burden of a single strain. Simultaneously, by utilizing a mixed-strain fermentation system combined with fermentation process regulation, the problems of high-concentration feedback inhibition of the intermediate metabolite arginine and the cytotoxicity of the final product butanediamine are effectively solved. Comparative experiments show that the butanediamine yield in a single-strain fermentation system is only 14.6~22.1 g / L, while the two-cell coupled system of this invention achieves a butanediamine yield of 40.9~45.2 g / L under the same raw material conditions, with a total sugar conversion rate of 0.22 g / g and a total sugar utilization rate exceeding 98%. Strain A at the fermentation endpoint OD... 600 It can still reach 42.3, while the OD of a single strain in the control group is lower. 600 Only 8.2. The above data fully demonstrates the superiority of this invention in overcoming feedback inhibition and product toxicity.

[0046] (2) Through dual-strain isolation and coupling design, the arginine synthesis metabolism of strain A is not directly inhibited or interfered with by the accumulation of butanediamine during the entire fermentation cycle. Its arginine synthesis pathway is not directly regulated by product accumulation, so strain A is always in a low-concentration environment of arginine, and the intracellular arginine concentration is continuously controlled below the feedback inhibition threshold, thus achieving sustained high arginine production. Furthermore, the synthesis process of butanediamine is entirely undertaken by strain B. To address the issue of strain B being directly exposed to the butanediamine environment, the dual T7 independent promoter expression system, combined with the optimization of fermentation process parameters such as IPTG concentration optimization (0.5 mM) and second-stage temperature synergistic regulation (35℃), achieves the matching of ARG and SpeC activities, controlling the peak intracellular butanediamine concentration below the tolerance threshold of strain B, and systematically reducing the cytotoxicity of butanediamine to strain B.

[0047] (3) Strong adaptability to raw materials: The system can be directly adapted to lignocellulose hydrolysate mixed with glucose and xylose without complicated separation treatment, and has good prospects for industrial application. Attached Figure Description

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0049] Figure 1 The graphs show the glucose consumption kinetics of different Escherichia coli genotypes in different culture media. Red dots (Glu) represent glucose consumption when glucose is used alone as a carbon source; yellow squares (Xylose) represent xylose consumption when xylose is used alone as a carbon source; red hollow circles (Glu(Glu+Xyl)) represent glucose consumption when glucose and xylose coexist; and yellow hollow squares (Xyl(Glu+Xyl)) represent xylose consumption when glucose and xylose coexist.

[0050] Figure 2 The plasmid map is for the recombinant plasmid pET28a-T7-ARG-T7-speC.

[0051] Figure 3 The time-history curves of key parameters during the two-cell coupled fermentation process are shown, including changes in arginine concentration, 1,4-butanediamine concentration, glucose concentration, and xylose concentration, as well as the total OD during fermentation. 600 change. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0053] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0054] Example 1: Construction of a butanediamine-transformed strain (denoted as strain B)

[0055] I. Construction of Xylose-Specific Utilization Strains BL21-ΔptsGΔglkΔmanZ

[0056] 1. Strain construction

[0057] Using CRISPR / Cas9 gene editing technology, the ptsG, glk, and manZ genes were knocked out sequentially. The specific procedures are as follows:

[0058] (1) Design and synthesis of targeting sgRNA

[0059] 20 nt sgRNA guide sequences were designed targeting the 50–150 bp region downstream of the start codon in the open reading frame (ORF) of the ptsG, glk, and manZ genes, respectively. Off-target effects were predicted using the online tool CRISPy-web, and the sgRNA with the highest specificity was screened. An adapter sequence compatible with the E. coli endogenous tRNA gene promoter (5'-GTCG-3') was added to the 5' end of the sgRNA sequence, and the complete sgRNA oligonucleotide chain was chemically synthesized by a commercial company. The nucleotide sequences of the ptsG, glk, and manZ genes are shown in SEQ ID NO. 1–3, and the corresponding 20 nt sgRNA guide sequences are shown in SEQ ID NO. 4–6, respectively.

[0060] (2) Construction of complete targeting fragments of ptsG, glk, and manZ genes

[0061] Using the *E. coli* BL21(DE3) genome as a template, overlapping extension PCR was used to amplify the upstream and downstream homologous arms (approximately 600 bp each) of the ptsG, glk, and manZ genes, as well as the central resistance selection marker (kanamycin resistance gene kan). Primers for the upstream and downstream homologous arms were designed to incorporate sequences complementary to both ends of the resistance marker. Three rounds of PCR fusion were used to obtain the complete targeting fragments of the ptsG, glk, and manZ genes. The targeting fragments were purified by gel electroporation for subsequent electroporation.

[0062] The primers used to construct the target fragment are shown in Table 1.

[0063] Table 1 Primers used to construct ptsG, glk, and manZ gene targeting fragments

[0064]

[0065] (3) Construction of CRISPR / Cas9 helper plasmids

[0066] The helper plasmid pCas9 (purchased from Addgene #42876), containing the Cas9 gene, λ-Red recombinase genes (gam, bet, exo), and a temperature-sensitive origin of replication (pSC101ts), was transformed into *E. coli* BL21(DE3) competent cells. Positive clones were screened on kanamycin-resistant plates at 30°C and 25 μg / mL. The three synthesized sgRNA oligonucleotide chains were annealed to form double strands and then cloned into the BsaI site of the pTarget plasmid (purchased from Addgene #62226), constructing recombinant plasmids pTarget-sgRNA-ptsG, pTarget-sgRNA-glk, and pTarget-sgRNA-manZ. Sequencing confirmed the correct sequences.

[0067] (4) Electroconversion and recombination screening

[0068] BL21(DE3) strain carrying the pCas9 plasmid was used to prepare electroporation competent cells. A pre-mixed targeting fragment (200 ng) and pTarget-sgRNA-ptsG plasmid (100 ng) were added, and after incubating on ice for 5 min, the cells were transferred to a 2 mm electroporation cuvette. The electroporation parameters were set as follows: 2.5 kV, 200 Ω, 25 μF. Immediately after electroporation, 1 mL of pre-chilled LB medium was added, and the cells were incubated at 30°C for 2 h. The cells were then plated on LB agar plates containing 25 μg / mL kanamycin and 0.5% L-arabinose (to induce λ-Red expression) and incubated upside down at 30°C for 24 h. Positive clones with correct sequencing were obtained by PCR identification.

[0069] (5) Elimination of resistance markers and helper plasmids

[0070] Positive clones, verified by sequencing, were inoculated into antibiotic-free LB broth containing 0.5% L-arabinose (which induces Cas9 and λ-Red expression) and cultured at 37°C with shaking for 12 h (the pCas9 plasmid was lost at 37°C due to a temperature-sensitive origin of replication). The clones were streaked onto antibiotic-free LB plates and incubated overnight at 37°C. Single colonies were picked and transferred to LB plates containing kanamycin (25 μg / mL) and streptomycin sulfate (40 μg / mL) respectively to screen for antibiotic-sensitive clones. Plasmid extraction further confirmed the elimination of both pCas9 and pTarget-sgRNA-ptsG, yielding the ptsG single-gene knockout strain BL21-ΔptsG.

[0071] (6) Using the obtained ptsG single-gene knockout strain BL21-ΔptsG as the starting strain, pCas9 plasmid was re-transformed into BL21-ΔptsG competent cells carrying pCas9. The complete glk gene targeting fragment (200 ng) and pTarget-sgRNA-glk (100 ng) were co-transformed into BL21-ΔptsG competent cells. After electroporation and resuscitation, the cells were plated on LB agar plates containing 34 μg / mL chloramphenicol and 0.5% L-arabinose and cultured at 30℃ for 24 h. Positive clones with correct sequencing were obtained by PCR identification. The ptsG and glk gene knockout strain BL21-ΔptsGΔglk was obtained by eliminating the resistance marker and helper plasmid.

[0072] (7) Then, obtain the ptsG and glk gene knockout strain BL21-ΔptsGΔglk as the starting strain, and refer to the experimental steps in step 5 to obtain the ptsG, glk and manZ gene knockout strain BL21-ΔptsGΔglkΔmanZ.

[0073] Strains BL21-ΔptsG, BL21-ΔptsGΔglk, and BL21-ΔptsGΔglkΔmanZ were stored in glycerol tubes at -80°C.

[0074] 2. Phenotypic Validation

[0075] After activating wild-type strains BL21(DE3), BL21-ΔptsG, BL21-ΔptsGΔglk, and BL21-ΔptsGΔglkΔmanZ, OD was regulated. 600 =0.1, inoculated at a 1% v / v rate into M9 basal medium containing 5 g / L glucose and 5 g / L xylose (M9 basal medium purchased from Sigma-Aldrich), and cultured at 37℃ with shaking at 200 rpm for 20–40 h. The residual sugar concentration and OD were then measured. 600 The control group was prepared using M9 basal medium containing only 5 g / L glucose and 5 g / L xylose. The results are as follows: Figure 1 As shown in Table 2, the BL21-ΔptsGΔglkΔmanZ strain, which knocks out the ptsG, glk, and manZ genes, does not consume glucose at all, has a xylose consumption rate of 100%, and exhibits optimal cell growth. It can achieve the target phenotype of glucose non-utilization and xylose high-efficiency utilization.

[0076] Table 2 Sugar consumption and growth of different strains in mixed sugar

[0077]

[0078] II. Construction of Xylose Metabolism Enhancement Strains

[0079] Using the BL21(DE3) genome as a template, the xylA gene (nucleotide sequence shown in SEQ ID NO. 7) encoding the xylose metabolism rate-limiting enzyme was amplified using primer pairs xylA-F and xylA-R; the xylB gene (nucleotide sequence shown in SEQ ID NO. 8) was amplified using primer pairs xylB-F and xylB-R. The ribosome binding site RBS sequence (nucleotide sequence shown in SEQ ID NO. 9) was introduced between xylA and xylB via overlap extension PCR, forming the xylA-RBS-xylB fusion fragment.

[0080] The xylA-RBS-xylB fusion fragment was ligated to the linearized constitutive T7 promoter-driven pET28a-T7 vector (purchased from Nanjing Herbal Source Biotechnology Co., Ltd.) using T4 ligase, transformed into DH5α, and the pET28a-T7-xylAB plasmid was extracted, verified by enzyme digestion, and sequenced to confirm its correctness. Using a heat shock method, the pET28a-T7-xylAB plasmid was transformed into competent cells of BL21-ΔptsGΔglkΔmanZ strain, plated on LB agar plates containing 25 μg / mL kanamycin, and cultured at 37°C for 12 h to obtain a xylose metabolism-enhanced strain. Transformation into the empty vector pET28a-T7 served as a control.

[0081] Seed cultures of the xylose metabolism-enhancing strain and the control strain were inoculated at a rate of 2% v / v into M9 basal medium containing 20 g / L xylose and cultured at 37°C with shaking at 200 rpm for 24 h. OD values ​​were then measured. 600 And xylose concentration. The results showed that the xylose consumption rate of the xylose-enhanced strain was increased by 52% compared with the empty vector control, and the final OD 600 It can reach 8.56.

[0082] The primers used to construct xylose metabolism-enhancing strains are shown in Table 3.

[0083] Table 3 Primers used for constructing xylose metabolism-enhancing strains

[0084]

[0085] III. Construction of the butanediamine-transformed strain (denoted as strain B)

[0086] 1. Construction of the dual T7 promoter co-expression plasmid pET28a-T7-ARG-T7-speC

[0087] (1) Construction of the ARG gene expression cassette: The bovine ARG gene sequence (SEQ ID NO. 10) optimized with E. coli codons was synthesized commercially and cloned into the pET28a vector. Using the synthesized plasmid as a template, primers ARG-F and ARG-R were designed, and the full-length ARG coding sequence was obtained by PCR amplification. The PCR product was double-digested with BamHI and HindIII and then ligated overnight at 16°C with the pET28a(+) vector backbone (retaining the N-terminal His-tag and the first T7 promoter) which had been double-digested with the same enzymes. The ligation product was transformed into E. coli DH5α competent cells, plated on LB plates containing 25 μg / mL kanamycin, and cultured at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the intermediate plasmid pET28a-T7-ARG was obtained after double enzyme digestion and sequencing confirmation.

[0088] (2) Amplification of the second T7 promoter and the speC gene expression cassette: Using the E. coli MG1655 genome as a template, the full-length speC gene (nucleotide sequence as shown in SEQ ID NO.11) was amplified by PCR using primers speC-F and speC-R. Simultaneously, using the pET28a(+) plasmid as a template, the expression cassette fragment containing the T7 promoter, RBS sequence, and transcription termination signal region was amplified using primers T7-F and T7-R. The two fragments were fused by overlap extension PCR to obtain the full-length T7-speC expression cassette. The product was digested with NheI and XhoI, and the digested products were recovered.

[0089] (3) Assembly of the dual expression cassette: The intermediate plasmid pET28a-T7-ARG was digested with NheI and XhoI to remove the original multiple cloning site region (approximately 50 bp), and the linearized vector backbone of approximately 5.2 kb was recovered. The linearized vector was mixed with the double-digested T7-speC expression cassette fragment at a molar ratio of 1:3, and Gibson Assembly premixed buffer (containing T5 exonuclease, Phusion DNA polymerase, and Taq ligase) was added. The mixture was incubated at 50°C for 1 h. The reaction product was transformed into DH5α competent cells, plated on LB plates containing 25 μg / mL kanamycin, and cultured overnight at 37°C. Single colonies were picked, plasmids were extracted, and the results were confirmed by NheI / XhoI double digestion and sequencing to obtain the dual T7 promoter co-expression plasmid pET28a-T7-ARG-T7-speC. The plasmid map is shown below. Figure 2 As shown.

[0090] (4) Construction of control plasmids: For subsequent functional verification, the empty vector control plasmid pET28a (without the insert fragment), the single gene expression plasmid pET28a-T7-ARG (i.e., the constructed intermediate plasmid pET28a-T7-ARG), and pET28a-T7-speC (the T7-speC expression cassette obtained is cloned into pET28a using NheI and XhoI) (double digestion of the vector to obtain a plasmid expressing only the speC gene) were constructed. All plasmids were sequenced to confirm their correctness and stored at -20℃ for later use.

[0091] The primers used to construct the dual T7 promoter co-expression plasmid pET28a-T7-ARG-T7-speC are shown in Table 4.

[0092] Table 4 Primers used for constructing the dual T7 promoter co-expression plasmid pET28a-T7-ARG-T7-speC

[0093]

[0094] 2. Heterologous expression of arginase gene ARG and ornithine decarboxylase gene speC and verification of their activities

[0095] The dual T7 promoter co-expression plasmid and each control plasmid were transformed into competent cells of strain BL21-ΔptsGΔglkΔmanZ or into competent cells of xylose metabolism-enhancing strains, respectively. Here, transformation into strain BL21-ΔptsGΔglkΔmanZ is used as an example. Positive clones were picked and inoculated into LB liquid medium containing 25 μg / mL kanamycin and cultured at 37°C until OD500. 600 ≈0.6, add 0.5 mM IPTG, and induce at 25℃ for 8 h. SDS-PAGE electrophoresis showed that specific protein bands appeared at the 35 kDa (ARG) and 75 kDa (speC) positions in the induced group.

[0096] Whole-cell catalysis experiment: The induced bacterial culture was centrifuged at 5000 rpm for 10 min at 4℃, the supernatant was discarded, and the bacterial cells were washed twice with pre-cooled 50 mM Tris-HCl buffer (pH 7.5), resuspended in the same buffer, and the OD was adjusted. 600Approximately 10 μL of L-arginine substrate was added, with a final concentration of 0.5 mM PLP and a final concentration of 2 mM MnSO4. The total catalytic reaction volume was 20 mL, and the reaction was carried out at 30 °C and 200 rpm for 2 h with shaking. After the reaction was completed, the supernatant was collected by centrifugation, and the butanediamine content was determined by HPLC. The results showed that the butanediamine production rate of the strain carrying the dual T7 promoter co-expression plasmid was 0.266 g / (L·h·OD), indicating that the synergistic catalytic activity of arginase and ornithine decarboxylase was good. The strain carrying the dual T7 promoter co-expression plasmid was designated as strain B.

[0097] 3. Investigation of the genetic stability of strain B

[0098] Strain B was passaged 50 times in antibiotic-free LB medium, with subculturing every 12 h. Samples were taken at generations 0, 20, 35, and 50, and plasmid loss rates were statistically analyzed on plates with and without kanamycin. Butanediamine conversion capacity was determined by shake-flask fermentation. The results showed that after 50 generations, the plasmid loss rate was approximately 18%, and the butanediamine yield decreased by 22%.

[0099] To further improve system stability, the pET28a-T7-ARG-T7-speC expression cassette was integrated into the attTn7 site of the B genome to construct an integrative strain without plasmids. Compared with plasmid-based strains, integrative strains do not require antibiotics to maintain selection pressure, and the yield decreases by less than 5% after 50 consecutive passages, which can significantly improve genetic stability and production cost advantages in industrial applications.

[0100] Example 2: Construction of an arginine-producing strain (denoted as strain A)

[0101] In this embodiment, Escherichia coli BL21(DE3) was used as the starting strain. Using CRISPR / Cas9 gene editing technology, a strain capable of efficiently synthesizing and secreting L-arginine using glucose as a single carbon source was constructed through systematic metabolic engineering (denoted as strain A).

[0102] 1. Degradation pathway blocking: Following the steps of knocking out the three genes ptsG, glk, and manZ in Example 1, using Escherichia coli BL21(DE3) as the starting strain, the arginine degradation-related genes speA, adiA, and astA were knocked out in sequence to obtain strain BL21-ARG1.

[0103] The nucleotide sequences of the speA, adiA, and astA genes are shown in SEQ ID NO. 12-14, and the corresponding 20 nt sgRNA guide sequences are shown in SEQ ID NO. 15-17. The primers used to construct the speA, adiA, and astA gene targeting fragments are shown in Table 5.

[0104] Table 5 Primers used to construct speA, adiA, and astA gene targeting fragments

[0105]

[0106] 2. Relief of feedback inhibition

[0107] Using BL21-ARG1 from step 1 as the starting strain, the argR gene was knocked out sequentially and the argJ recombinant fragment was integrated using the CRISPR / Cas9 system.

[0108] (1) Knock out the global negative regulator argR

[0109] A 20 nt sgRNA guide sequence (SEQ ID NO.19) was designed targeting the 80 bp region downstream of the ORF start codon of the argR gene (SEQ ID NO.18), cloned into the pTarget vector to construct the pTarget-sgRNA-argR plasmid (the construction process of the pTarget-sgRNA-argR plasmid is described in step (3) of the strain construction in Example 1), and sequenced for verification. Using the BL21(DE3) genome as a template, the upstream and downstream homologous arms of the argR gene (approximately 600 bp each) were amplified, and the kan gene was used as a selection marker to fuse them into a targeting fragment via OE-PCR. After transforming the pCas9 plasmid into BL21-ARG1 competent cells, the targeting fragment (200 ng) and the pTarget-argR-sgRNA plasmid (100 ng) were co-electroporated into the competent cells (2.5 kV, 200 Ω, 25 μF). The cells were incubated in LB medium at 30°C for 2 h, plated on LB agar containing 25 μg / mL kanamycin and 0.5% L-arabinose, and cultured at 30°C for 24 h. Single colonies were picked and colony PCR was performed using primers for detecting the argR gene. The amplification product of positive clones changed from approximately 1.2 kb in the wild type to approximately 0.8 kb. After sequencing confirmation, the cells were passaged in antibiotic-free LB medium at 37°C to eliminate the plasmid, yielding strain BL21-ARG1ΔargR.

[0110] (2) Integrating the argJ gene and replacing the function of argE

[0111] The argJ gene (approximately 1.2 kb, SEQ ID NO.20) was amplified using the Corynebacterium glutamicum genome as a template, and the Pprc constitutive strong promoter (approximately 300 bp, SEQ ID NO.21) was amplified using the pSTV28 vector as a template. The two gene sequences were then fused into the Pprc-argJ expression unit via OE-PCR.

[0112] Construction of pTarget-lacI-sgRNA plasmid targeting the lacI site: A 20nt sgRNA guide sequence (SEQ ID NO. 22) was designed targeting the 120 bp region downstream of the ORF start codon of the lacI gene in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 23, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 24. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added for ligation overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-lacI-sgRNA plasmid.

[0113] The upstream and downstream homologous arms of the lacI site (each approximately 600 bp) were amplified and fused with the aforementioned Pprc-argJ expression unit and chloramphenicol resistance gene to form a targeting fragment. This targeting fragment and pTarget-lacI-sgRNA targeting the lacI site were co-electroplated into BL21-ARG1ΔargR competent cells carrying pCas9. Chloramphenicol-resistant clones were screened and verified by PCR using the outer primer of lacI (wild-type approximately 1.0 kb, integrated approximately 2.2 kb). After sequencing confirmation, the plasmid was eliminated to obtain strain BL21-ARG2.

[0114] BL21-ARG2 was inoculated into a fermentation medium containing 40 g / L glucose (fermentation medium: glucose 40 g / L, xylose 20 g / L, (NH4)2SO4 4 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 6 g / L, MgSO4·7H2O 1.5 g / L, yeast extract 2 g / L, ZnSO4·7H2O 0.17 g / L, FeSO4·7H2O 0.02 g / L, MnSO4·H2O 0.01 g / L, V B1 V B3 V B5 And D-biotin 0.002 g / L. After sterilization, the pH was adjusted to 7.0 with ammonia water, and the mixture was incubated at 37℃ and 200 rpm for 28 h. HPLC analysis showed that the arginine yield reached 1.9 g / L, confirming that the feedback inhibition was effectively relieved.

[0115] The primers used in step 2 of Example 2 are shown in Table 6.

[0116] Table 6 Primers used in step 2 of Example 2

[0117]

[0118] 3. Enhancement of the synthetic pathway

[0119] Using BL21-ARG2 obtained in step 2 as the starting strain, a four-step enhanced synthesis pathway was developed.

[0120] Step 1: Integrate the argCJBDF operon at the yghX site

[0121] Using the Corynebacterium glutamicum genome as a template, the argC (SEQ ID NO.25), argB (SEQ ID NO.26), argD (SEQ ID NO.27), and argF (SEQ ID NO.28) genes were amplified respectively. They were then fused into an operon by OE-PCR in the order of argC-argB-argD-argF and placed downstream of the Pprc promoter (SEQ ID NO.21) to obtain the Pprc-argCJBDF recombinant fragment.

[0122] Construction of pTarget-yghX-sgRNA plasmid targeting the yghX site: A 20 nt sgRNA guide sequence (SEQ ID NO. 29) was designed targeting the ORF region of the yghX pseudogene site in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 30, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 31. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-yghX-sgRNA plasmid.

[0123] Homologous arms upstream and downstream of the yghX pseudogene locus were amplified. The Pprc-argCJBDF recombinant fragment was fused with a kan resistance marker to form a targeting fragment, which, along with pTarget-yghX-sgRNA, was electroporated into BL21-ARG2 competent cells carrying pCas9. Positive clones were screened, and PCR verification was performed using the outer primers of yghX (approximately 1.0 kb in wild type and approximately 6.3 kb after integration). Sequencing confirmed the result, yielding strain BL21-ARG3. Fermentation verification showed that arginine production increased to 12.7 g / L.

[0124] Step 2: Integrate the argGH operator at the ygaY site

[0125] Using the genome of Corynebacterium glutamicum as a template, the argG (SEQ ID NO.32) and argH (SEQ ID NO.33) genes were amplified, respectively, and fused in the argG-argH sequence by OE-PCR and placed downstream of the Pprc promoter to obtain the Pprc-argGH recombinant fragment.

[0126] Construction of pTarget-ygaY-sgRNA plasmid targeting the ygaY site: A 20 nt sgRNA guide sequence (SEQ ID NO. 34) was designed targeting the ORF region of the ygaY pseudogene site in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 35, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 36. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-ygaY-sgRNA plasmid.

[0127] Homologous arms upstream and downstream of the ygaY pseudogene locus were amplified, and the Pprc-argGH recombinant fragment was fused with a kan resistance marker to form a targeting fragment. The pTarget-ygaY-sgRNA plasmid, along with the targeting fragment, was electroporated into BL21-ARG3 competent cells carrying pCas9. PCR verification (wild-type approximately 0.9 kb, integrated approximately 3.6 kb) and sequencing confirmed the formation of strain BL21-ARG4. In this strain, heterologous argJ replaced the function of the natural argE gene, constructing an acetyl recycling pathway. Fermentation verification showed that the byproduct acetic acid decreased from 5.4 g / L to 1.3 g / L, while arginine production increased to 13.1 g / L.

[0128] Step 3: Integrate pyrAA / pyrAB at the yciV site

[0129] Using the Bacillus subtilis F126 genome as a template, the pyrAA (SEQ ID NO.37) and pyrAB-E949G (SEQ ID NO.38) genes were amplified, fused by OE-PCR in the pyrAA-pyrAB sequence, and placed downstream of the Pprc promoter to obtain the Pprc-pyrAA / pyrAB recombinant fragment.

[0130] Construction of pTarget-yciV-sgRNA plasmid targeting the yciV site: A 20 nt sgRNA guide sequence (SEQ ID NO. 39) was designed targeting the ORF region of the yciV pseudogene site in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 40, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 41. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-yciV-sgRNA plasmid.

[0131] Homologous arms (approximately 600 bp each) upstream and downstream of the yciV pseudogene locus were amplified, and the Pprc-pyrAA / pyrAB recombinant fragment was fused with a kan resistance marker to form a targeting fragment. The pTarget-yciV-sgRNA plasmid, along with the targeting fragment, was electroporated into BL21-ARG4 competent cells carrying pCas9. PCR and sequencing confirmed the formation of strain BL21-ARG5. Fermentation validation showed that arginine production increased to 15.9 g / L after carbamoyl phosphate enhancement.

[0132] Step 4: Integration of pntAB at the yjiP site

[0133] The pntAB gene (SEQ ID NO.42) was amplified using the BL21(DE3) genome as a template and placed downstream of the Pprc promoter to obtain the Pprc-pntAB recombinant fragment.

[0134] Construction of pTarget-yjiP-sgRNA plasmid targeting the yjiP site: A 20 nt sgRNA guide sequence (SEQ ID NO. 43) was designed targeting the ORF region of the yjiP pseudogene site in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 44, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 45. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-yjiP-sgRNA plasmid.

[0135] Homologous arms (approximately 600 bp each) upstream and downstream of the yjiP pseudogene locus were amplified, and the Pprc-pntAB recombinant fragment was fused with a kan resistance marker to form a targeting fragment. The pTarget-yjiP-sgRNA plasmid, along with the targeting fragment, was electroporated into BL21-ARG5 competent cells carrying pCas9. PCR and sequencing confirmed the formation of strain BL21-ARG6. Fermentation analysis showed that arginine production reached 17.7 g / L after NADPH enhancement.

[0136] The primer information used in step 3 is shown in Table 7.

[0137] Table 7 Primer information used in step 3 of Example 2

[0138]

[0139] 4. Dynamic control of TCA carbon flow

[0140] Using BL21-ARG6 as the starting strain, the natural promoter of the sucA gene was replaced with the growth-dependent promoter PrpsT (SEQ ID NO.46) to achieve autonomous switching of metabolic flux from the exponential phase to the stationary phase.

[0141] Construction of pTarget-sucA-sgRNA plasmid targeting the sucA promoter region: A 20 nt sgRNA guide sequence (SEQ ID NO. 47) was designed targeting the sucA gene promoter region in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 48, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 49. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-sucA-sgRNA plasmid.

[0142] The sucA gene was amplified by extending approximately 600 bp upstream (homologous arm 1) and downstream (homologous arm 2). Using the artificially synthesized pUC57-PrpsT plasmid containing the PrpsT promoter sequence as a template, the PrpsT promoter was amplified. Homologous arm 1, PrpsT, kan marker, and homologous arm 2 were sequentially fused using OE-PCR to form a targeting fragment of approximately 2.2 kb. The pTarget-sucA-sgRNA plasmid and the targeting fragment were co-electroplated into BL21-ARG6 competent cells carrying pCas9. Positive clones were screened, and verification was performed using primers outside the sucA promoter region (approximately 0.4 kb for wild-type and approximately 0.8 kb after replacement). After sequencing confirmation, the plasmid was eliminated, yielding strain BL21-ARG9.

[0143] BL21-ARG9 was inoculated into a fermentation medium containing 40 g / L glucose (fermentation medium: glucose 40 g / L, xylose 20 g / L, (NH4)2SO4 4 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 6 g / L, MgSO4·7H2O 1.5 g / L, yeast extract 2 g / L, ZnSO4·7H2O 0.17 g / L, FeSO4·7H2O 0.02 g / L, MnSO4·H2O 0.01 g / L, V B1 V B3 V B5 Add 0.002 g / L of D-biotin. After sterilization, adjust the pH to 7.0 with ammonia water. Fermentation was carried out at 37℃ and 200 rpm. Total RNA was extracted from bacterial samples at 4 h and 12 h of fermentation, reverse transcribed into cDNA, and then qRT-PCR was performed using sucA-specific primers. The results showed that the expression level of sucA mRNA at 12 h was 95.7% lower than that of wild type. At the same time, bacterial cells were sonicated and the α-KGDH enzyme activity (NADH production rate at 340 nm) was measured. The results showed that the enzyme activity decreased by 63.2% within 4-12 h, confirming that the PrpsT promoter achieved autonomous switching of metabolic flux from the exponential phase to the stationary phase. Shake flask fermentation verified that the arginine yield reached 21.8 g / L, which was 23.2% higher than that of BL21-ARG6, and OD 600 The value was 6.1, and the growth of the strain was not significantly affected.

[0144] The primer information used in step 4 is shown in Table 8.

[0145] Table 8 Primer information used in step 4 of Example 2

[0146]

[0147] 5. Enhanced external drainage system

[0148] Using BL21-ARG9 as the starting strain, the lysE gene from Corynebacterium glutamicum was integrated into the ilvG site. Specifically, the lysE gene (SEQ ID NO.50) was amplified using the Corynebacterium glutamicum genome as a template and fused with the Pprc promoter (SEQ ID NO.21) to form the Pprc-lysE recombinant fragment.

[0149] Construction of pTarget-ilvG-sgRNA plasmid targeting the ilvG site: A 20 nt sgRNA guide sequence (SEQ ID NO. 51) was designed targeting the ORF region of the ilvG gene site in the *E. coli* BL21(DE3) genome. Off-target effects were predicted using the online tool CRISPy-web to confirm its specificity. The complementary oligonucleotide chain of the guide sequence (containing sticky ends compatible with the BsaI restriction site of the pTarget vector) was chemically synthesized commercially. The forward oligonucleotide chain sequence is shown in SEQ ID NO. 52, and the reverse oligonucleotide chain sequence is shown in SEQ ID NO. 53. The pTarget plasmid (Addgene #62226) was digested with BsaI restriction endonuclease at 37°C for 2 h, and the linearized vector backbone was recovered by gel electrophoresis. The two synthesized sgRNA oligonucleotide chains were heated at 95°C for 5 min and then slowly annealed to room temperature to form a double-stranded DNA fragment. The annealed product was mixed with the linearized pTarget vector backbone at a molar ratio of 5:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 50 μg / mL spectinomycin, then incubated at 37°C for 12 h. Single colonies were picked, plasmids were extracted, and the recombinant plasmid was sequenced using sequencing primers to confirm the correct insertion of the sgRNA guide sequence, thus obtaining the pTarget-ilvG-sgRNA plasmid.

[0150] The upstream and downstream homologous arms of the ilvG site (approximately 600 bp each) were amplified and fused with the Pprc-lysE recombinant fragment and the kan marker to form a targeting fragment of approximately 2.4 kb. The pTarget-ilvG-sgRNA plasmid and the targeting fragment were co-electroplated into BL21-ARG9 competent cells carrying pCas9. Positive clones were screened and verified by PCR using the outer primers of ilvG (wild-type approximately 0.6 kb, integrated approximately 2.4 kb). After sequencing confirmation, the plasmid was eliminated, yielding strain BL21-ARG12, designated as strain A.

[0151] Strain A was inoculated into a fermentation medium containing 40 g / L glucose (fermentation medium: glucose 40 g / L, xylose 20 g / L, (NH4)2SO4 4 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 6 g / L, MgSO4·7H2O 1.5 g / L, yeast extract 2 g / L, ZnSO4·7H2O 0.17 g / L, FeSO4·7H2O 0.02 g / L, MnSO4·H2O 0.01 g / L, V B1 V B3 V B5And D-biotin 0.002 g / L. After sterilization, the pH was adjusted to 7.0 with ammonia water, and the mixture was incubated at 37℃ and 200 rpm for 28 h. HPLC analysis showed that the arginine yield reached 28.3 g / L, OD 600 The value was 6.3, which was 29.8% higher than that of BL21-ARG9, indicating that lysE overexpression effectively enhanced arginine efflux and further relieved feedback inhibition.

[0152] Example 3: Optimization of fermentation conditions and determination of key parameter ranges for butanediamine production by two-cell coupled fermentation

[0153] Example 1 constructed a butanediamine-transforming strain (denoted as strain B), and Example 2 constructed an arginine-producing strain (denoted as strain A). This example, based on the previous construction of strains A and B, utilizes two-cell coupled fermentation of strains A and B to produce butanediamine, systematically optimizing fermentation conditions and key parameters in the two-cell coupled system that may affect butanediamine yield, production efficiency, and toxicity inhibition effect.

[0154] 1. Exploration of single-factor conditions

[0155] A 5 L fermenter (working volume 3 L) was used. Fermentation medium: glucose 40 g / L, xylose 20 g / L, (NH4)2SO4 4 g / L, KH2PO4 3 g / L, Na2HPO4·12H2O 6 g / L, MgSO4·7H2O 1.5 g / L, yeast extract 2 g / L, ZnSO4·7H2O 0.17 g / L, FeSO4·7H2O 0.02 g / L, MnSO4·H2O 0.01 g / L, V B1 V B3 V B5 Add 0.002 g / L of D-biotin. After sterilization, adjust the pH to 7.0 with ammonia.

[0156] Phase 1: After activating strain A, a single colony was picked and inoculated into LB liquid medium, and cultured with shaking until the logarithmic growth phase to obtain a seed culture. The seed culture was inoculated into a 5 L fermenter at an inoculation rate of 10% v / v, at a temperature of 37℃ and dissolved oxygen of 30%, with glucose continuously added to maintain a concentration of 5-10 g / L. After 20 h of fermentation, the arginine concentration reached 35 g / L.

[0157] The subsequent second-stage optimization used the following initial baseline conditions: a second-stage temperature of 35℃, a second-stage dissolved oxygen level of 30%, an IPTG concentration of 0.2 mM, and an inoculation ratio of strain A to strain B of 5:1. Under these baseline conditions, the effects of each factor were investigated one by one.

[0158] (1) Second stage temperature optimization

[0159] Second stage: Seed culture of strain B was inoculated at a ratio of strain A:strain B = 5:1 (cell biomass), and 0.2 mMIPTG was added. The temperature was controlled at 30℃, 33℃, 35℃, 37℃, and 39℃, respectively, and dissolved oxygen was adjusted to 30%. Glucose feeding was stopped, and xylose was fed in at 500 g / L to maintain a xylose concentration of 5-10 g / L. Fermentation continued for 28 h, for a total of 48 h. The yield of butanediamine, arginine residue, and growth of strain B were investigated at different temperatures in the second stage.

[0160] The results are shown in Table 9. When the temperature in the second stage was controlled at 35℃, the yield of butanediamine was the highest at 40.9 g / L, the arginine residue was the lowest (<0.5 g / L), and strain B grew well. Below 33℃, the activities of ARG and SpeC enzymes were insufficient, and the arginine conversion efficiency decreased; above 37℃, the high temperature exacerbated the membrane damage of butanediamine to strain B, the relative membrane damage index decreased, and the cells lysed prematurely. Therefore, the optimal temperature range is 33℃~37℃, preferably 35℃.

[0161] Table 9. Effects of different second-stage temperatures on fermentation performance

[0162]

[0163] (2) Second stage dissolved oxygen optimization

[0164] Second stage: Seed culture of strain B was inoculated at a ratio of strain A:strain B = 5:1 (cell biomass), 0.2 mMIPTG was added, and the temperature was controlled at 35℃. Dissolved oxygen was adjusted to 20%, 30%, 40%, 50%, and 60%, respectively. Glucose feeding was stopped, and xylose was fed in at 500 g / L to maintain a xylose concentration of 5-10 g / L. Fermentation continued for 28 h, for a total of 48 h. The effect of different dissolved oxygen control levels in the second stage on fermentation performance was investigated.

[0165] Table 10 Effects of different dissolved oxygen levels on fermentation performance

[0166]

[0167] The results are shown in Table 10. When dissolved oxygen was controlled at 30%–50%, both butanediamine production and xylose consumption were optimal, with the lowest accumulation of the byproduct acetic acid. When dissolved oxygen was below 30%, insufficient oxygen supply limited the growth of strain B and resulted in incomplete xylose utilization; when dissolved oxygen was above 50%, high dissolved oxygen may cause partial oxidative stress, slightly inhibiting the activity of strain B. Therefore, the optimal dissolved oxygen range is 30%–50%.

[0168] (3) Optimization of IPTG concentration

[0169] Second stage: Seed culture of strain B was inoculated at a ratio of strain A:strain B = 5:1 (cell biomass). 0.2, 0.5, 0.8, 1.0, and 1.5 mM IPTG were added respectively. The temperature was controlled at 35℃, dissolved oxygen was adjusted to 30%, glucose feeding was stopped, and 500 g / L xylose was started to maintain a xylose concentration of 5-10 g / L. Fermentation continued for 28 h, for a total of 48 h. The effects of different IPTG concentrations on the matching of ARG and SpeC enzyme activities and the yield of butanediamine were investigated.

[0170] Table 11 Effects of different IPTG concentrations on the activity of the two enzymes and the yield of butanediamine.

[0171]

[0172] The results are shown in Table 11. When the IPTG concentration was 0.5 mM, the activity ratio of ARG to SpeC was closest to 1:3 (the theoretical optimal matching value), the intracellular succinyldiamine peak was the lowest (45 mg / g DCW), and the succinyldiamine yield was the highest (42.8 g / L). When the IPTG concentration was too low (≤0.2 mM), the expression of both enzymes was insufficient, resulting in low arginine conversion efficiency; when the IPTG concentration was too high (≥1.0 mM), SpeC overexpression led to the intracellular accumulation rate of succinyldiamine exceeding the efflux capacity, resulting in increased cytotoxicity and decreased yield. Therefore, the optimal IPTG concentration was 0.5 mM.

[0173] (4) Optimization of inoculation ratio (strain A: strain B)

[0174] Second stage: Seed culture of strain B was inoculated with strain A: strain B at ratios of 1:1, 2:1, 5:1, 8:1, and 10:1 (cell biomass). 0.2 mM IPTG was added to each ratio, the temperature was controlled at 35℃, dissolved oxygen was adjusted to 30%, glucose feeding was stopped, and 500 g / L xylose was fed in to maintain a xylose concentration of 5-10 g / L. Fermentation continued for 28 h, for a total of 48 h, to investigate the effect of different inoculation ratios on fermentation synergistic efficiency.

[0175] Table 12 Effect of different inoculum ratios on fermentation performance

[0176]

[0177] The results are shown in Table 12. When the inoculation ratio was 5:1, the two strains showed optimal synergistic growth, with the highest yield of butanediamine (41.2 g / L) and extremely low arginine residue. When the ratio was too low (≤2:1), there was an excessive amount of strain B, leading to rapid xylose consumption but insufficient arginine supply; when the ratio was too high (≥10:1), the number of strain B strains was insufficient, resulting in delayed arginine conversion and subsequent feedback inhibition. Therefore, the optimal inoculation ratio was 5:1, with a preferred range of 5:1 to 8:1.

[0178] 2. Precise Optimization Using Multi-Factor Response Surface Method (RSM)

[0179] Based on the parameter range determined by single-factor optimization (second-stage temperature 33℃~37℃, second-stage dissolved oxygen 30%~50%, IPTG 0.5~0.8 mM, inoculation ratio 5:1~8:1), a four-factor, three-level response surface analysis was conducted using a Box-Behnken design, with butanediamine yield as the response value.

[0180] The optimal process parameters predicted by the model were: temperature 34.8℃, dissolved oxygen 44.5%, IPTG 0.52 mM, and inoculum ratio 5.2:1, with a predicted maximum butanediamine yield of 46.1 g / L. For ease of practical operation, the optimal conditions were adjusted to: temperature 35℃, dissolved oxygen 45%, IPTG 0.5 mM, and inoculum ratio 5:1. The butanediamine yields were 44.8, 45.5, and 45.2 g / L, with an average of 45.2 ± 0.3 g / L. The total sugar conversion rate was 0.22 ± 0.01 g / g, arginine residue < 0.5 g / L, xylose residue < 0.5 g / L, and total sugar utilization > 98%. This indicates that the process parameters have good repeatability and are stable and reliable. The good agreement with the predicted values ​​proves the reliability of the response surface model.

[0181] 3. Analysis of the program curves of key parameters in typical fermentation processes

[0182] Under the above optimal parameter conditions, the program curves of key parameters during fermentation were analyzed, and the results are as follows: Figure 3 As shown (second stage temperature 35℃, second stage dissolved oxygen 45%, IPTG 0.5 mM, inoculation ratio 5:1).

[0183] Sugar concentration changes: In the first stage (0~20 h), glucose was rapidly consumed by strain A, and fed-batch feeding maintained the glucose concentration at 5~15 g / L; in the second stage (after 20 h), the xylose concentration began to decrease, and at 48 h, the xylose residue was <0.5 g / L.

[0184] Arginine accumulation and transformation: At the end of the first stage (20 h), the arginine concentration reached 35 g / L; after the inoculation of strain B in the second stage, the arginine concentration rapidly decreased to <8 g / L within 2-4 h, and then remained at a low level of 1-3 g / L, proving that the immediate consumption of arginine by strain B effectively relieved the feedback inhibition.

[0185] Butyldiamine formation: After the start of the second stage, the concentration of butyldiamine continued to rise, reaching 45.2 g / L after 48 h. The formation rate of butyldiamine was highly matched with the consumption rate of arginine throughout the process, indicating that the activities of ARG and SpeC were well matched.

[0186] Cell growth: Total OD during fermentation 600 In the second stage, the total OD value stabilized at around 65 (the mixed biomass of strains A and B), and the fermentation endpoint (48 h) was [value missing]. 600 The value was 50.8, indicating that neither strain was significantly inhibited by butanediamine toxicity and both were in good growth condition.

[0187] The above data shows that the optimized parameters can achieve the ideal two-phase distribution state of "low concentration maintenance of arginine and high concentration accumulation of butanediamine", thereby simultaneously obtaining high yield, high conversion rate and low toxicity inhibition.

[0188] Example 4: Comparative experiment of two-cell coupling system and single-strain fermentation and verification of inhibition relief effect

[0189] To verify the effectiveness of the dual-cell coupling system of this invention in relieving arginine feedback inhibition and butanediamine cytotoxicity, the following comparative experiment was designed.

[0190] 1. Experimental Group A (the dual-cell coupling system of this invention): fermented for 48 h in a 5 L fermenter under the optimized fermentation conditions of Example 3.

[0191] 2. Control Group B (Single-Strain Full-Pathway Fermentation): Using wild-type *Escherichia coli* BL21(DE3) as the starting strain, key genes in the arginine synthesis pathway (including feedback-relieved mutant argA, argB-H, lysE, and argO) as well as ARG and speC genes were overexpressed in this strain to construct a single engineered strain that completes the entire conversion from a mixed carbon source of glucose and xylose to butanediamine. Fermentation conditions were as optimized in Example 3, employing simultaneous inoculation and mixed sugar feeding.

[0192] 3. Control group C (double bacterial coupling): using strain A and strain B', strain B' is strain B that does not express ARG and speC genes, and accumulates naturally only after strain A secretes arginine.

[0193] 4. Control group D (double-strain coupling): using strain A' and strain B, but strain A' is strain A that does not express the lysE efflux protein gene.

[0194] Because two different microorganisms, strain A (or strain A') and strain B (strain B'), coexist in a two-cell coupled fermentation system, conventional spectrophotometry can only determine the total OD of the mixed bacterial solution. 600 The values ​​do not distinguish the individual biomass of each strain. To quantify the cell concentration of the two strains separately, this invention uses the selective plate count (CFU) method. The specific operating steps are as follows:

[0195] At each key time point of fermentation (including the end of the first stage and every 4–6 hours after inoculation of strain B in the second stage), 1 mL of sample was aseptically taken from the fermenter. The bacterial culture was then serially diluted 10-fold with sterile physiological saline (to a final concentration of 10). -5 ~10 -7 100 μL of bacterial suspension at each dilution was spread onto two types of solid plates: ① LB agar plates containing 25 ug / mL kanamycin (strain B carries the kanamycin resistance plasmid pET28a-T7-ARG-T7-speC and can grow normally on this plate, while strain A does not have kanamycin resistance and cannot grow), and ② LB agar plates without antibiotics (total viable count). Three replicates were prepared for each dilution, and the plates were incubated upside down at 37°C for 24 h after spreading. The colony count was counted on each plate (plates with colony counts between 30 and 300 were selected for counting). The concentration of strain B and the total viable count were calculated using the formula: strain concentration (CFU / mL) = colony count × dilution factor × 10. The concentration of strain A was obtained by subtracting the concentration of strain B from the total viable count.

[0196] Meanwhile, another sample of fermentation broth was appropriately diluted (to reduce OD). 600 The total OD value was measured using a UV-Vis spectrophotometer (within the range of 0.2 to 0.8). 600 Values. The CFU values ​​of strains A and B at each time point were compared with the corresponding total OD values. 600 A standard curve between the values ​​allows for rapid estimation of the biomass of the two strains in subsequent experiments. The OD values ​​of strain A (or strain A') and strain B (or strain B') reported in this example... 600 The values ​​are all obtained by conversion based on the standard curve established by the CFU method mentioned above, and can truly reflect their growth status in the mixed fermentation system.

[0197] The results of the four comparative experiments are shown in Table 13. In control group B, the single strain needs to simultaneously undertake multiple metabolic tasks such as mixed sugar utilization, arginine synthesis, and arginine conversion. The feedback inhibition of arginine (21.5 g / L residual) on argA by high concentration leads to severe restriction of strain growth (OD). 600 In control group C, despite the use of two-strain coupling, strain B' lacked arginine conversion ability, and arginine continued to accumulate to 52.3 g / L. The feedback inhibition from strain A was not relieved, resulting in a butanediamine yield of only 3.8 g / L. In control group D, strain A' lacked arginine efflux protein, and the excessively high intracellular arginine concentration led to feedback inhibition by argA, thus inhibiting the growth of strain A (OD). 600 Only 22.4 g / L), butanediamine production was only 22.8 g / L.

[0198] Data from experimental group A showed that the synergistic effect of arginine efflux from strain A and immediate transformation by strain B maintained the arginine concentration in the fermentation broth at an extremely low level (<0.5 g / L), completely eliminating feedback inhibition, and strain A maintained a good growth state (OD). 600 =42.3), butanediamine production reached 45.2 g / L. Xylose residue <0.5 g / L, and total sugar utilization exceeded 98%. The above results demonstrate that the dual design of overexpressing the arginine efflux protein gene in strain A and expressing ARG and speC in strain B has a significant synergistic effect, and the technical effect of the present invention cannot be achieved by using either strategy alone.

[0199] Table 13 Comparison of butanediamine yield, arginine residue, and strain growth in different fermentation systems

[0200]

[0201] Example 5: Application of Butanediamine Production from Real Lignocellulose Hydrolysate via Two-Cell Coupled Fermentation

[0202] Corn stalks were sterilized at 121°C for 20 min using 2% dilute sulfuric acid. The supernatant was collected by filtration, and the pH was adjusted to 5.5 with Ca(OH)2. The gypsum precipitate was removed by centrifugation, and the supernatant was detoxified by adsorption with 2% activated carbon before filtration for sterilization. HPLC analysis showed glucose 28.5 g / L, xylose 12.3 g / L, acetic acid 1.8 g / L, and furfural 0.15 g / L. Carbon sources were added to bring the glucose concentration to 40 g / L and the xylose concentration to 20 g / L. The same nitrogen source and inorganic salts as in Example 3 were added, and fermentation was carried out according to the optimized process and parameters of Example 3. Finally, the yield of butanediamine was determined to be 41.2 g / L, and the sugar conversion rate was 0.20 g / g; the yield of the undetoxified hydrolysate control group was only 28.0 g / L.

[0203] This invention provides a method for producing butanediamine using two-cell coupled fermentation. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for producing butanediamine using two-cell coupled fermentation, characterized in that, Includes the following steps: (1) First stage: The seed culture of arginine-producing strain A was inoculated into a mixed carbon source medium containing glucose and xylose and fermented until the arginine concentration in the fermentation broth reached 30~35 g / L. During the fermentation process, glucose was added to maintain the glucose concentration at 5~15 g / L. (2) Second stage: According to the cell biomass ratio of arginine production strain A to butylene diamine conversion strain B = 5:1~8:1, continue to inoculate the seed liquid of butylene diamine conversion strain B into the fermentation broth of step (1), and stop the flow of glucose, and instead flow xylose to maintain the xylose concentration at 5~10 g / L, and continue fermentation to convert arginine into butylene diamine; The arginine-producing strain A was obtained by using Escherichia coli BL21(DE3) as the starting strain, knocking out arginine degradation-related genes speA, adiA, and astA, knocking out the global negative regulator argR, integrating argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, pntAB, and lysE genes, and replacing the natural promoter of the sucA gene with the PrpsT promoter. The butanediamine-transformed strain B was obtained by knocking out the ptsG, glk, and manZ genes and overexpressing the arginase gene ARG and the ornithine decarboxylase gene speC, using Escherichia coli BL21(DE3) as the starting strain.

2. The method according to claim 1, characterized in that, The butanediamine-transformed strain B also overexpresses the xylose isomerase encoding gene xylA and the xylose kinase encoding gene xylB.

3. The method according to claim 1, characterized in that, The nucleotide sequences of the arginine degradation-related genes speA, adiA, and astA are shown in SEQ ID NO. 12-14, the nucleotide sequence of the global negative regulator argR is shown in SEQ ID NO. 18, and the nucleotide sequences of the argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, pntAB, and lysE genes are shown in SEQ ID NO. 20, SEQ ID NO. 25-28, SEQ ID NO. 32-33, SEQ ID NO. 42, and SEQ ID NO. 50, respectively; the nucleotide sequence of the PrpsT promoter is shown in SEQ ID NO.

46.

4. The method according to claim 1, characterized in that, The nucleotide sequences of the ptsG, glk, and manZ genes are shown in SEQ ID NO. 1-3, and the nucleotide sequences of the arginase ARG and ornithine decarboxylase speC are shown in SEQ ID NO. 10-11.

5. The method according to claim 1 or 2, characterized in that, The nucleotide sequences of the xylose isomerase encoding gene xylA and the xylose kinase encoding gene xylB are shown in SEQ ID NO.7~8, respectively.

6. The method according to claim 1, characterized in that, The argJ gene is driven by the Pprc promoter and integrates into the lacI site; the argC, argB, argD, and argF genes, after sequential fusion, are driven by the Pprc promoter and integrate into the yghX pseudogene site; the argG and argH genes, after sequential fusion, are driven by the Pprc promoter and integrate into the ygaY pseudogene site; the pyrAA and pyrAB genes, after sequential fusion, are driven by the Pprc promoter and integrate into the yciV pseudogene site; the pntAB gene is driven by the Pprc promoter and integrates into the yjiP pseudogene site; and the lysE gene is driven by the Pprc promoter and integrates into the ilvG site.

7. The method according to claim 6, characterized in that, The nucleotide sequence of the Pprc promoter is shown in SEQ ID NO.

21.

8. The method according to claim 1, characterized in that, In step (2), 0.5~0.8 mM IPTG is added to the fermentation broth from step (1), and the temperature of the second stage is controlled at 33℃~37℃ and the dissolved oxygen at 30%~50%.

9. An arginine-producing strain for the two-cell coupled fermentation production of butanediamine according to claim 1, characterized in that, The arginine-producing strain was obtained by using Escherichia coli BL21(DE3) as the starting strain, knocking out arginine degradation-related genes speA, adiA, and astA, knocking out the global negative regulator argR, integrating argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, and lysE genes, and replacing the natural promoter of the sucA gene with the PrpsT promoter. The nucleotide sequences of the arginine degradation-related genes speA, adiA, and astA are shown in SEQ ID NO. 12-14, the nucleotide sequence of the global negative regulator argR is shown in SEQ ID NO. 18, and the nucleotide sequences of the genes argJ, argC, argB, argD, argF, argG, argH, pyrAA, pyrAB, pntAB, and lysE are shown in SEQ ID NO. 20, SEQ ID NO. 25-28, SEQ ID NO. 32-33, SEQ ID NO. 42, and SEQ ID NO. 50, respectively; the nucleotide sequence of the PrpsT promoter is shown in SEQ ID NO.

46.

10. A butanediamine-transforming strain for the two-cell coupled fermentation production of butanediamine according to claim 1, characterized in that, It was obtained by knocking out the ptsG, glk, and manZ genes and overexpressing the arginase gene ARG and the ornithine decarboxylase gene speC, using Escherichia coli BL21(DE3) as the starting strain. The nucleotide sequences of the ptsG, glk, and manZ genes are shown in SEQ ID NO. 1-3, and the nucleotide sequences of the arginase ARG and ornithine decarboxylase speC are shown in SEQ ID NO. 10-11.

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