An engineered bacterium for producing isoleucine, a construction method thereof and application thereof

Abstract

The application discloses an engineered bacterium for producing isoleucine, a construction method and application thereof, and relates to the technical field of biotechnology. The construction method comprises the following steps: transforming a coding gene of a fusion protein into a chassis strain to construct a recombinant strain; the recombinant strain is the engineered bacterium; and the fusion protein comprises Bcd protein, Linker and IlvD protein sequentially connected from an N terminal to a C terminal. The application combines the concept of synthetic biology with metabolic engineering, designs Linkers with different lengths and optimizes the connection sequence, constructs an IlvD-Bcd fusion protein, realizes spatial proximity of downstream double enzymes and substrate channelization, and thus significantly improves the synthesis flux of isoleucine, thereby providing a new technical scheme for efficient biological manufacturing of isoleucine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an engineered bacterium for producing isoleucine, its construction method, and its application. Background Technology

[0002] Isoleucine, as a branched-chain amino acid, has wide applications in the pharmaceutical, food, and feed industries. Microbial fermentation is the main method for producing isoleucine; however, traditional metabolic engineering often focuses on the overexpression or knockout of single genes, lacking systematic optimization of the overall pathway throughput. In particular, the low efficiency of intermediate product transfer in downstream continuous reaction steps limits further yield increases.

[0003] The development of synthetic biology has provided new tools and ideas for the fine regulation of metabolic pathways. Fusion protein technology, as one of the important tools of synthetic biology, connects multiple enzymes at the protein level through flexible linkers, making them spatially adjacent to each other and forming "metabolic channels," which can significantly improve the overall kinetic efficiency of continuous reactions.

[0004] In the production of isoleucine via E. coli fermentation, a critical technical bottleneck remains: the low synergistic efficiency of the downstream dual enzymes IlvD (dihydroxy acid dehydratase) and Bcd (branched-chain amino acid dehydrogenase). Specifically, the intermediate 2-keto-3-methylvaleric acid (KMV) catalyzed by IlvD needs to diffuse to the active site of Bcd to be further converted into isoleucine. However, the two enzymes exist independently within the cell, and the diffusion loss of the intermediate and competition from side reactions severely restrict the overall throughput. This spatial isolation leads to low transfer efficiency, limiting further high-yield isoleucine production. This invention aims to develop a novel engineered bacterium for isoleucine production, thereby providing technical support for the industrial-scale, high-efficiency production of isoleucine. Summary of the Invention

[0005] The purpose of this invention is to provide an engineered bacterium for producing isoleucine, its construction method, and its application, thereby addressing the problems existing in the prior art. This engineered bacterium can effectively increase isoleucine yield, thus providing technical support for the industrial-scale, efficient production of isoleucine.

[0006] Addressing the challenges in isoleucine biomanufacturing, constructing a fusion protein of IlvD and Bcd and applying it to enhance downstream metabolic pathways presents a promising solution. Fusion protein technology, through flexible linkers, spatially proximates the two enzymes, enabling the direct capture and conversion of KMV generated by IlvD by Bcd, reducing diffusion losses of intermediate products, and thus achieving highly efficient synergistic improvement in downstream throughput of the isoleucine biosynthesis pathway. Addressing the technical bottleneck of low synergistic efficiency of downstream two enzymes in the isoleucine biosynthesis pathway, this invention combines synthetic biology concepts with metabolic engineering. By designing flexible linkers of different lengths and optimizing the connection sequence, an IlvD-Bcd fusion protein is constructed, achieving spatial proximate proximity and substrate channelization of the downstream two enzymes, thereby significantly improving the biosynthetic throughput of isoleucine and providing a new technical solution for the efficient biomanufacturing of isoleucine.

[0007] Based on this, the present invention provides the following solution: This invention provides a fusion protein for increasing isoleucine production, the fusion protein comprising a Bcd protein, a Linker, and an IlvD protein connected sequentially from the N-terminus to the C-terminus; The amino acid sequence of the Bcd protein is shown in SEQ ID NO.1; The amino acid sequence of the IlvD protein is shown in SEQ ID NO.3; The Linker is (GGGGS) n Where n is 4.

[0008] The present invention also provides a gene encoding the above-mentioned fusion protein.

[0009] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.

[0010] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0011] The present invention also provides the application of the above-mentioned encoding gene or recombinant expression vector in constructing recombinant strains that produce high levels of isoleucine.

[0012] The present invention also provides a method for constructing an engineered bacterium that produces isoleucine, comprising the step of transforming the above-mentioned encoding gene into a chassis strain to construct a recombinant strain; the recombinant strain is the engineered bacterium.

[0013] Furthermore, the strain in the chassis is Escherichia coli, Corynebacterium, or Bacillus.

[0014] Furthermore, the *Escherichia coli* is *Escherichia coli* Y315; The *E. coli* Y315 strain was developed using *E. coli* MG1655 as the starting strain. This was achieved by knocking out the ilvA, rhtA, tdcG, tdcE, ΔldhA, and ΔbrnQ genes, and overexpressing ilvA. Gene, ilvE gene, ilvIH 1 gene, ilvIH The 2-gene, ilvC-ec gene, and ilvD gene were constructed. The ilvA The nucleotide sequence of the gene is shown in SEQ ID NO.8; The ilvIH The nucleotide sequence of gene 1 is shown in SEQ ID NO.13; The ilvIH The nucleotide sequence of gene 2 is shown in SEQ ID NO.14.

[0015] The present invention also provides an engineered bacterium constructed according to the above-described construction method.

[0016] The present invention also provides a method for producing isoleucine by fermentation, comprising the step of fermenting and culturing the above-mentioned engineered bacteria to obtain isoleucine.

[0017] The present invention discloses the following technical effects: This invention develops a fusion protein that can enhance the synergistic efficiency of downstream dual enzymes in the isoleucine synthesis pathway, showing great potential for increasing isoleucine yield. By optimizing the linker length (20 aa) and linkage sequence (Bcd-IlvD), this invention achieves efficient transfer of the intermediate 2-keto-3-methylvaleric acid (KMV) generated by IlvD to Bcd, significantly reducing diffusion loss and side reaction competition. Building upon this, this invention further incorporates multiple rounds of metabolic engineering modifications, including strong promoter-driven upstream ilvA, increased ilvIH copy number, and optimized NADPH supply. Simultaneously, the fermentation process of the constructed high-yield isoleucine strain was optimized, establishing a high-efficiency fermentation route for isoleucine synthesis from *E. coli*. Ultimately, this resulted in a shake-flask level isoleucine yield of 13.0 g / L, providing a new technical means and approach for the green production of branched-chain amino acids such as isoleucine.

[0018] This invention successfully verifies the effectiveness of fusion protein metabolic channel strategies in enhancing continuous enzymatic reactions, providing a new paradigm and reference for the spatial organization optimization of multi-enzyme cascade reactions in the field of metabolic engineering. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 OD of recombinant strains constructed using linkers of different lengths 600 (A) and (B) statistics of isoleucine production; Figure 2 OD of recombinant strains constructed using different ligation sequences 600 (A) and (B) Statistical chart of isoleucine production. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, all experimental techniques and methods employed in this invention follow generally accepted technical paths and operating procedures in the field. Specifically, all experimental operations, including but not limited to molecular biology experiments, microbial culture and identification, and biochemical analysis, are performed in accordance with standard procedures found in authoritative textbooks and industry guidelines. For example, experimental steps without clearly defined operating conditions are typically performed under standard laboratory conditions, detailed in classic works such as *Molecular Cloning: A Laboratory Manual (3rd Edition)* (Science Press) and *Microbiology Experiments (4th Edition)* (Higher Education Press). Alternatively, the official recommendations provided by the manufacturers of the reagents, instruments, and consumables used in the experiment may be followed. Regarding the selection of experimental materials and reagents, unless otherwise specified in this embodiment, all biological materials (such as bacterial strains and cell lines), chemical reagents (including but not limited to buffer solutions, enzyme preparations, and antibodies), and experimental consumables (such as culture dishes, centrifuge tubes, and pipette tips) used are procured through legal and compliant commercial channels to ensure their quality meets experimental requirements and industry standards.

[0027] For the quantitative analysis experiments in the examples, to ensure the accuracy and reliability of the data, each experiment was independently repeated three times to eliminate the influence of random errors. Finally, the data obtained from the three repeated experiments were statistically analyzed, and the arithmetic mean was taken as the final result of that experimental group. Subsequent data processing and result interpretation were then based on this result. This process strictly adheres to the basic principles of scientific experimentation, aiming to improve the credibility and repeatability of the experimental conclusions.

[0028] Terminology Explanation: Dihydroxy-acid dehydratase (IlvD) is a key enzyme in the isoleucine biosynthesis pathway, catalyzing the dehydration of 2,3-dihydroxy-3-methylvaleric acid to 2-keto-3-methylvaleric acid (KMV). IlvD belongs to the dehydratase family, and its active site contains an iron-sulfur cluster ([4Fe-4S]), which catalyzes the elimination of hydroxyl groups in the substrate molecule to form a double bond, providing a direct precursor for subsequent transamination / dehydrogenation reactions. In its native state, IlvD exists as a homodimer, and its catalytic efficiency directly affects the downstream product supply flux. In this invention, IlvD, as a component of a fusion protein, is linked to the downstream enzyme Bcd via a flexible linker, achieving efficient delivery of the intermediate product.

[0029] Branched-chain amino acid dehydrogenase (Bcd) is a key enzyme catalyzing the final step of isoleucine synthesis, using NADH as a cofactor to reduce and amination 2-keto-3-methylvaleric acid (KMV) to isoleucine. Bcd belongs to the amino acid dehydrogenase family and exhibits high specificity for branched-chain keto acid substrates; the conformational change of its active site is closely related to the cofactor binding state. In high-yield isoleucine strains, the catalytic efficiency of Bcd is a key factor determining downstream flux. In this invention, Bcd, as a component of a fusion protein, efficiently captures KMV generated by IlvD through spatial proximity, significantly improving the overall kinetic efficiency of the continuous reaction.

[0030] (GGGGS) n A flexible linker is a short peptide linker sequence composed of glycine (Gly) and serine (Ser) repeating units, used to link IlvD and Bcd at the protein level. Glycine, with its hydrogen side chain, possesses extremely high conformational freedom, giving the linker excellent flexibility. The hydroxyl side chain of serine is hydrophilic, helping to maintain the extended conformation of the linker in an aqueous environment and reducing non-specific interactions with the functional domains on both sides. By adjusting the number of repeating units n (in this invention, n = 2, 3, 4, 5, corresponding to linker lengths of 10, 15, 20, and 25 amino acids), the spatial distance between IlvD and Bcd can be precisely controlled, thereby screening for the conformation with optimal metabolic channel efficiency. Experiments in this invention demonstrate that a linker with n = 4 (20 amino acids) enables both enzymes to maintain their correct folding while forming an efficient substrate channel, achieving the optimal KMV delivery efficiency.

[0031] The term "metabolic channeling" used in this invention refers to the phenomenon where intermediate products in a continuous enzymatic reaction are directly transferred from the active site of the upstream enzyme to the downstream enzyme through spatial organization optimization, avoiding their diffusion into the cytoplasmic medium. In natural metabolic pathways, intermediate products must undergo free diffusion to be captured by downstream enzymes, a process that involves diffusion loss, competitive consumption, and the risk of side reactions. This invention, by constructing an IlvD-Bcd fusion protein, shortens the spatial distance between the two enzymes to the nanoscale, enabling the KMV generated by IlvD to be directly captured and converted by Bcd, significantly improving the overall efficiency of the continuous reaction. The strength of the metabolic channeling effect can be indirectly evaluated by the accumulation of intermediate product KMV: the lower the KMV accumulation, the stronger the channeling effect and the smaller the carbon flux loss. In this invention, the Bcd-20aa-IlvD fusion protein exhibits the lowest KMV accumulation and the highest isoleucine production, confirming its excellent metabolic channeling effect.

[0032] Chassis strains are fundamental host strains in synthetic biology and bioengineering, characterized by clear genetic backgrounds and stable performance. They are essentially the "chassis" of a cell factory capable of carrying exogenous genes and metabolic pathways. Primarily serving as universal hosts for expressing exogenous proteins, synthesizing drugs, chemicals, and other target products, they achieve efficient and controllable biosynthesis through standardized modification, forming the core foundation of synthetic biology and fermentation engineering.

[0033] The pTargetF plasmid involved in this invention has been disclosed in the literature “Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015;81(7):2506-2514”.

[0034] The amino acid sequence of the Bcd protein is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; the amino acid sequence of the IlvD protein is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4; the amino acid sequence of the Bcd-20aa-IlvD fusion protein is shown in SEQ ID NO.5, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.6; the nucleotide sequence of the threonine dehydratase gene (ilvA) is shown in SEQ ID NO.7; the threonine dehydratase mutant gene (ilvA)... The nucleotide sequences of the following genes are shown in SEQ ID NO. 8: threonine efflux protein gene (rhtA); serine dehydratase gene (tdcG); branched-chain amino acid transaminase gene (ilvE); acetylhydroxyl synthase gene (ilvIH); and acetylhydroxyl synthase mutant 1 gene (ilvIH). 1) The nucleotide sequence is shown in SEQ ID NO.13; acetylhydroxyl synthase mutant 2 gene (ilvIH) 2) The nucleotide sequence of the gene is shown in SEQ ID NO.14; the nucleotide sequence of the 2-ketobutyrate-formate lyase gene (tdcE) is shown in SEQ ID NO.15; the nucleotide sequence of the lactate dehydrogenase gene (ldhA) is shown in SEQ ID NO.16; the nucleotide sequence of the branched-chain amino acid transporter gene (brnQ) is shown in SEQ ID NO.17; the nucleotide sequence of the ketool acid reductase gene (ilvC-ec) is shown in SEQ ID NO.18; and the nucleotide sequence of the dihydroxy acid dehydratase gene (ilvD) is shown in SEQ ID NO.19.

[0035] The primer information involved in this invention is shown in Table 1.

[0036] Table 1 Primer Information Example 1 Construction method of strain Y315: 1. Knock out the ilvA gene in MG1655 Using i11 / i12 as primers and pTargetF plasmid as template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-ilvA plasmid that had successfully replaced N20.

[0037] Using i13 / i14 as primers and the genome of wild-type Escherichia coli MG1655 (hereinafter referred to as MG1655 genome) as template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis to obtain a fragment of approximately 200 bp in size. Then, the fragment was recovered by gel electrophoresis to obtain the upstream repair fragment.

[0038] Using i15 / i16 as primers and the MG1655 genome as a template, PCR amplification was performed to obtain a fragment of approximately 200 bp, which was then recovered by gel extraction to obtain the downstream repair fragment.

[0039] Using i13 / i16 as primers and the recovered upstream and downstream repair fragments as templates, overlap extension PCR amplification was performed to ligate the upstream and downstream fragments. The PCR products were detected by agarose gel electrophoresis, and the fragments were excised and recovered to obtain a DornorΔilvA fragment of approximately 400 bp.

[0040] The DornorΔilvA fragment and the constructed pTargetF-ilvA plasmid were electroporated into pre-prepared wild-type Escherichia coli MG1655 electroporated competent cells. 800 μL of LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. 200 μL of the incubation medium was then evenly spread on spectinomycin-containing solid LB plates and incubated upside down at 37°C overnight. After single colonies grew, the cells were passaged. Once the passaged cells showed obvious growth, PCR amplification and identification were performed on the single colonies using i17 / i18 primers. 2 μL of the product was taken for agarose gel electrophoresis, and a 500 bp target band was obtained and sent to the company for sequencing. Finally, the MG1655 strain with ilvA knocked out was obtained, denoted as MG1655ΔilvA.

[0041] 2. Overexpression of PJ23119-ilvA Construction of gene-engineered bacteria Using i19 / i20 as primers and pTargetF plasmid as template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-rhtA plasmid that had successfully replaced N20.

[0042] Using i21 / i22 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain an upstream repair fragment containing the PJ23119 promoter.

[0043] Using i23 / i24 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 1500 bp. The fragment was then recovered from the gel to obtain a midstream repair fragment containing the PJ23119 promoter.

[0044] Using i25 / i26 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 300 bp. The fragment was then recovered from the gel to obtain the third repair fragment.

[0045] Using i27 / i28 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the fourth repair fragment.

[0046] Using i21 / i26 as primers, and templates containing upstream repair fragments, midstream repair fragments, third repair fragments, and fourth repair fragments of the PJ23119 promoter, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding fragments of approximately 2200 bp in size. The fragments were then recovered from the gel to obtain the complete repair fragments.

[0047] The repair fragment and the constructed pTargetF-rhtA plasmid were electroporated into pre-prepared competent E. coli MG1655ΔilvA cells. 800 μL of LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. 200 μL of the incubation medium was then evenly spread onto spectinomycin-containing solid LB plates and incubated overnight at 37°C. After single colonies appeared, the cells were passaged. Once the passaged cells showed clear growth, PCR amplification was performed on the single colonies using i21 / i40 primers. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 2200 bp target band, which was then sent to the company for sequencing. The MG1655 strain with successfully knocked-in ilvA was obtained, designated MG1655ΔilvAΔrhtA::PJ23119-ilvA. .

[0048] 3. Overexpression of ilvE Using i79 / i80 primers and pTargetF plasmid as a template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-tdcG plasmid that had successfully replaced N20.

[0049] Using i81 / i82 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the upstream repair fragment.

[0050] Using i83 / i84 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 1000 bp. The fragment was then recovered from the gel to obtain the ilvE midstream repair fragment containing the Ptrc promoter.

[0051] Using i85 / i86 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the downstream repair fragment.

[0052] Using i81 / i86 as primers, and upstream repair fragment, midstream repair fragment containing the Ptrc promoter and downstream repair fragment as templates, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 1500 bp. The fragment was then recovered from the gel to obtain the complete repair fragment.

[0053] The complete repair fragment and the constructed pTargetF-tdcG plasmid were electroporated into the pre-prepared E. coli MG1655ΔilvAΔrhtA::PJ23119-ilvA. In competent cells, 800 μL of LB medium was added and the cells were incubated at 37°C and 200 rpm for 1 h. 200 μL of the incubation medium was then evenly spread onto spectinomycin-containing solid LB plates and incubated overnight at 37°C. After single colonies appeared, the cells were passaged. Once the passaged cells showed clear growth, PCR amplification was performed on the single colonies using i81 / i90 primers for identification. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 1600 bp target band, which was then sent to the company for sequencing. The MG1655 strain with successful ilvE knock-in was obtained, designated MG1655ΔilvAΔrhtA::PJ23119-ilvA. ΔtdcG::Ptrc-ilvE.

[0054] 4. Overexpression of 3 copies of ilvIH (1) The first time that ilvIH on the genome was mutated to ilvIH 1 Using C58 / C59 primers and pTargetF plasmid as a template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-ilvIH plasmid that had successfully replaced N20.

[0055] Using i358 / C61 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the upstream repair fragment.

[0056] Using C60 / i359 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 500 bp. The fragment was then recovered from the gel to obtain the downstream repair fragment.

[0057] Using i358 / i359 as primers and upstream and downstream repair fragments as templates, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 400 bp. The fragment was then recovered from the gel to obtain the complete repair fragment.

[0058] The complete repair fragment and the constructed pTargetF-ilvIH plasmid were electroporated into the pre-prepared E. coli MG1655ΔilvAΔrhtA::PJ23119-ilvA. In competent cells of ΔtdcG::Ptrc-ilvE, 800 μL of LB medium was added, and the cells were revived at 37°C and 200 rpm for 1 h. 200 μL of the revival medium was then evenly spread onto spectinomycin-containing solid LB agar plates and incubated overnight at 37°C. After single colonies grew, the cells were passaged. Once the passaged cells showed clear growth, PCR amplification was performed on the single colonies using i358 / i348 primers for identification. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 1000 bp target band, which was then sent to the company for sequencing. The final result showed that ilvIH was mutated to ilvIH. 1 strain of MG1655, denoted as MG1655ΔilvAΔrhtA::PJ23119-ilvA ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH 1.

[0059] (2) Second insertion of ilvIH 2 Using i151 / i152 primers and pTargetF plasmid as template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-tdcE1 plasmid that had successfully replaced N20.

[0060] Using i153 / i154 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 160 bp. The fragment was then recovered from the gel to obtain the upstream repair fragment.

[0061] Using i155 / i158 as primers, with ilvIH Using the two genes as templates, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 2400 bp. The fragment was then recovered from the gel to obtain the midstream repair fragment.

[0062] Using i159 / i160 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the downstream repair fragment.

[0063] Using i153 / i160 as primers and upstream, midstream, and downstream repair fragments as templates, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 2700 bp. The fragment was then recovered from the gel to obtain the complete repair fragment.

[0064] The complete repair fragment and the constructed pTargetF-tdcE1 plasmid were electroporated into the previously prepared E. coli MG1655ΔilvAΔrhtA::PJ23119-ilvA from the previous step. ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH In 1 competent cell, 800 μL of LB medium was added and the cells were revived at 37°C and 200 rpm for 1 h. 200 μL of the revival medium was then evenly spread onto a spectinomycin-containing solid LB agar plate and incubated overnight at 37°C. After single colonies appeared, the cells were passaged. Once the passaged cells showed clear growth, PCR amplification was performed on the single colonies using i149 / i150 primers for identification. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 2900 bp target band, which was then sent to the company for sequencing. The result was a successful insertion of ilvIH. 2 strains of MG1655, denoted as MG1655ΔilvAΔrhtA::PJ23119-ilvA ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH 1ΔtdcE::Ptrc-ilvIH 2.

[0065] (3) Third insertion of ilvIH 1 Using i411 / i412 as primers and pTargetF plasmid as template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-ldhA plasmid that had successfully replaced N20.

[0066] Using i413 / i414 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 300 bp. The fragment was then recovered from the gel to obtain the upstream repair fragment.

[0067] Using i385 / i416 as primers, with ilvIH Using gene 1 as a template, PCR amplification was performed. The PCR product was detected by agarose gel electrophoresis, yielding a fragment of approximately 2400 bp. The fragment was then recovered from the gel to obtain the midstream repair fragment.

[0068] Using i417 / i418 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 350 bp. The fragment was then recovered from the gel to obtain the downstream repair fragment.

[0069] Using i413 / i418 as primers and upstream, midstream, and downstream repair fragments as templates, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 400 bp. The fragment was then recovered from the gel to obtain the complete repair fragment.

[0070] The complete repair fragment and the constructed pTargetF-ldhA plasmid were electroporated into the pre-prepared E. coli MG1655ΔilvAΔrhtA::PJ23119-ilvA. ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH 1ΔtdcE::Ptrc-ilvIH In two competent cells, 800 μL of LB medium was added, and the cells were revived at 37°C and 200 rpm for 1 h. 200 μL of the revival medium was then evenly spread onto a spectinomycin-containing solid LB agar plate, inverted, and incubated overnight at 37°C. After single colonies grew, the cells were passaged. Once the passaged cells showed clear growth, PCR amplification was performed on the single colonies using i419 / i420 primers for identification. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 3100 bp target band, which was then sent to the company for sequencing. The successfully inserted ilvIH was finally obtained. The engineered strain 1 is represented as MG1655ΔilvAΔrhtA::PJ23119-ilvA ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH 1ΔtdcE::Ptrc-ilvIH 2ΔldhA::ilvIH 1.

[0071] 5. Overexpression of ilvC-ec on the genome Using AY3302 / AY3303 as primers and pTargetF plasmid as template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, revealing a linear and mutated pTargetF plasmid of approximately 2100 bp. The plasmid was then recovered from the gel and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using the Q5 point mutation kit. The resulting mixture was transformed into *E. coli* DH5α competent cells. 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing. The resulting plasmid successfully replaced N... 20 The pTargetF-brnQ plasmid.

[0072] Using AY3304 / AY3305 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. This fragment was then recovered from the gel, yielding a fragment containing P. J23119 Upstream repair fragment of the promoter.

[0073] Using AY3288 / AY3307 as primers and the MG1655 genome as a template, PCR was performed to obtain a 1600 bp ilvC-ec midstream fragment.

[0074] Using AY3308 / AY3309 as primers and the MG1655 genome as a template, PCR amplification was performed to obtain a 200 bp downstream repair fragment.

[0075] Using the upstream repair fragment, the midstream ilvC-ec fragment, and the downstream repair fragment as templates, and AY3304 / AY3309 as primers, PCR amplification was performed to obtain the ilvC-ec repair fragment.

[0076] The ilvC-ec repair fragment and the constructed pTargetF-brnQ plasmid were electroporated into a pre-prepared *E. coli* MG1655ΔilvAΔrhtA::PJ23119-ilvA ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH ΔtdcE::Ptrc-ilvIH ΔldhA::ilvIH In competent cells, 800 μL of LB medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h. 200 μL of the incubation medium was then evenly spread onto spectinomycin-containing solid LB plates, which were inverted and incubated overnight at 37°C. After single colonies appeared, the cells were passaged. Once the passaged cells showed clear growth, PCR amplification was performed on the single colonies using AY3310 / AY3311 primers for identification. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 2200 bp target band, which was then sent to the company for sequencing. The strain overexpressing ilvC-ec was finally obtained, designated MG1655ΔilvAΔrhtA::P J23119 -ilvA ΔtdcG::P trc -ilvEΔilvIH::ilvIH ΔtdcE::P trc -ilvIH ΔldhA::ilvIH ΔbrnQ::ilvC-ec.

[0077] 6. Overexpression of ilvD on the genome Using i69 / i70 primers and pTargetF plasmid as a template, PCR amplification was performed. 20 μL of the PCR product was analyzed by agarose gel electrophoresis, yielding a linear and mutated pTargetF plasmid of approximately 2100 bp. The gel was then recovered and eluted with 10 μL of ultrapure water. The PCR product was then subjected to a KLD reaction using a Q5 point mutation kit. The resulting mixture was transferred into *E. coli* DH5α competent cells, and 50 μL of the culture was plated onto LB agar plates containing spectinomycin and incubated upside down at 37°C until single colonies appeared. Transformants were passaged on LB agar plates containing spectinomycin, and colony PCR was performed using primers TF-F / TF-R. A 600 bp band was considered a positive clone and sent for sequencing, ultimately yielding the pTargetF-tdcE2 plasmid that had successfully replaced N20.

[0078] Using i61 / i62 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the upstream repair fragment.

[0079] Using i63 / i64 as primers and the genome of strain i135 preserved in the laboratory as a template, PCR amplification was performed. The PCR product was detected by agarose gel electrophoresis, and a fragment of approximately 2100 bp was obtained. Then, the fragment was recovered by gel electrophoresis to obtain the midstream repair fragment.

[0080] Using i65 / i66 as primers and the MG1655 genome as a template, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 200 bp. The fragment was then recovered from the gel to obtain the downstream repair fragment.

[0081] Using i61 / i66 as primers and upstream, midstream, and downstream repair fragments as templates, PCR amplification was performed. The PCR products were detected by agarose gel electrophoresis, yielding a fragment of approximately 2400 bp. The fragment was then recovered from the gel to obtain the complete repair fragment.

[0082] The complete repair fragment and the pTargetF-tdcE2 plasmid constructed in the laboratory using i69 / i70 primers were electroporated into the pre-prepared E. coli MG1655ΔilvAΔrhtA::PJ23119-ilvA from the previous step. ΔtdcG::Ptrc-ilvEΔilvIH::ilvIH ΔtdcE::Ptrc-ilvIH ΔldhA::ilvIH In competent cells of ΔbrnQ::ilvC-ec, 800 μL of LB medium was added, and the cells were revived at 37°C and 200 rpm for 1 h. 200 μL of the revival medium was then evenly spread onto a spectinomycin-containing solid LB agar plate, inverted, and incubated overnight at 37°C. After single colonies grew, the cells were subcultured. Once the subcultured cells showed clear growth, PCR amplification was performed on the subcultured single colonies using i149 / i150 primers. 2 μL of the product was analyzed by agarose gel electrophoresis, yielding a 2900 bp target band, which was then sent to the company for sequencing. The strain that successfully inserted ilvD was finally obtained, designated MG1655ΔilvAΔrhtA::P J23119 -ilvA ΔtdcG::P trc -ilvEΔilvIH::ilvIH ΔtdcE::P trc -ilvIH ΔldhA::ilvIH ΔbrnQ::ilvC-ec-ilvD, renamed to Y315.

[0083] Example 2 Construction method of Y383 plasmid: With P BB Using the promoter fragment as a template and AY3334 / AY3317 as primers, PCR was performed to obtain P with homologous arms. BB Excerpt.

[0084] Using AY3334 / AY3319 as primers, and P with homologous arms... BB Fragments and ilvIH Using gene 1 as a template, PCR was performed to obtain a gene carrying the P gene. BB promoter and upstream and downstream homologous arms ilvIH 1 segment.

[0085] Using the bcd gene as a template and AY3362 / AY3361 as primers, the bcd gene fragment was obtained by PCR reaction.

[0086] Using AY3344 / AY3339 as primers and a bcd gene fragment containing both the bcd promoter and terminator as a template, a PCR reaction was performed to obtain a gene containing P. J23105 BCD fragments of promoters, terminators, and upstream and downstream homologous arms.

[0087] Using pSW11 plasmid as a template and AY3336 / AY3342 as primers, a linearized vector backbone was obtained through PCR reaction.

[0088] Take 3 μL of the fragment (including those containing P) BB promoter and upstream and downstream homologous arms ilvIH 1 fragment and those with P J23105 The promoter, terminator, and bcd fragments of upstream and downstream homologous arms were used. 2 μL of linearized vector backbone was ligated using CE ligase in a PCR instrument at 50°C for 15 min. The ligation product was then aspirated and mixed, and transformed into 100 μL of DH5α competent cells. The cells were incubated on ice for 30 min, heat-shocked at 42°C for 45 s, and then placed on ice for 2 min. 800 μL of LB medium was added, and the cells were incubated at 37°C at 200 rpm for recovery. 100 μL of the recovery buffer was plated onto Amp plates. The cells were incubated overnight at 37°C. Transformants were passaged on LB plates containing ampicillin for 1 h. Colony PCR was performed using primers AY3338 / AY3147. A 3900 bp band was considered a positive clone and sent for sequencing. The resulting cells were successfully ligated with ilvIH. Y383 plasmid containing the 1 and bcd genes.

[0089] Example 3 In the isoleucine biosynthesis pathway, IlvD (dihydroxyacid dehydratase) and Bcd (branched-chain amino acid dehydrogenase) are key enzymes catalyzing downstream sequential reactions. IlvD catalyzes the dehydration of 2,3-dihydroxy-3-methylvaleric acid to 2-keto-3-methylvaleric acid (KMV), and subsequently, Bcd catalyzes the conversion of KMV to the final product isoleucine. This step consumes one molecule of NADPH. These two enzymes catalyze sequential reactions, and the intermediate product KMV needs to diffuse from the active site of IlvD to the active site of Bcd. In their native state, the two enzymes exist independently within the cell. KMV requires cytoplasmic diffusion to be captured by Bcd. During this process, some KMV may be competitively utilized by other enzymes, diluted in the cytoplasm, or even flow into side reactions, resulting in carbon flux loss and decreased pathway efficiency.

[0090] Fusion protein technology uses flexible linkers to connect two enzymes at the protein level, placing them spatially close to each other and creating a "metabolic channel" effect. This effect allows intermediate products produced by the upstream enzyme to be directly captured and converted by the downstream enzyme, reducing diffusion losses and competition for intermediate products, thereby improving the overall kinetic efficiency of the continuous reaction. Key parameters in fusion protein design include the linker length and the connection order of the two enzymes. A linker that is too short may cause interference between the two enzymes, affecting their correct folding; a linker that is too long may not effectively constrain spatial distance, weakening the channel effect. The connection order should follow the metabolic flow, placing the upstream enzyme at the N-terminus and the downstream enzyme at the C-terminus, ensuring that the intermediate product transport pathway aligns with the metabolic direction.

[0091] I. Construction of recombinant strains using linkers of different lengths As a key element connecting two enzymes, the linker's length directly affects the spatial conformation of the fusion protein and the relative positions of the active sites of the two enzymes. A linker that is too short may cause interference between the two enzymes, affecting their correct folding; a linker that is too long may fail to effectively constrain the spatial distance between the two enzymes, weakening the channel effect. This section of the experiment aims to systematically investigate the effects of flexible linkers of different lengths (10 aa, 15 aa, 20 aa, 25 aa) connecting IlvD and Bcd on isoleucine synthesis, to screen for the optimal linker length, and to provide a basis for constructing efficient fusion proteins.

[0092] This invention uses a flexible linker sequence (GGGGS) rich in glycine and serine. n This sequence exhibits good flexibility and hydrophilicity, minimizing its impact on the structures of enzymes on both sides. Four linkers of different lengths were designed: L10: (GGGGS)2, 10 amino acids in length; L15: (GGGGS)3, 15 amino acids in length; L20: (GGGGS)4, 20 amino acids in length; and L25: (GGGGS)5, 25 amino acids in length. A linker with 0 amino acids was used as a control.

[0093] Using plasmid Y383 as a template and primers AY3359 / AY3342 as primers, a linearized plasmid backbone containing the genes of the upstream and downstream homologous arms of 0 aalinker was obtained by PCR amplification.

[0094] Using plasmid Y383 as a template and primers AY3364 / AY3342 as primers, a linearized plasmid backbone containing 10 aalinker homologous arm genes was obtained by PCR amplification.

[0095] Using plasmid Y383 as a template and primers AY3415 / AY3342 as primers, a linearized plasmid backbone containing 15 aalinker and 20 aa linker homologous arm genes was obtained by PCR amplification.

[0096] Using plasmid Y383 as a template and primers AY3416 / AY3342 as primers, a linearized plasmid backbone containing the 25 aalinker homologous arm gene was obtained by PCR amplification.

[0097] All linearized plasmid backbones contain the ilvIH and bcd genes.

[0098] Using AY3376 / AY3339 as primers and the MG1655 genome as a template, PCR amplification was performed to obtain samples containing bcd homology arms and plasmid homology arms. ilvD Gene fragment 1.

[0099] Using AY3373 / AY3339 as primers and the MG1655 genome as a template, PCR amplification was performed to obtain homologous arms containing 10 aalinker / 15 aa linker arms and plasmid homologous arms. ilvD Gene fragment 2.

[0100] Using AY3366 / AY3339 as primers and the MG1655 genome as a template, PCR amplification was performed to obtain homologous arms containing 20 aalinker / 25 aa linker arms and plasmid homologous arms. ilvD Gene fragment 3.

[0101] Connecting linearized plasmid backbone 1 and ilvD Gene fragment 1 was used to obtain ligation product 1.

[0102] Connecting linearized plasmid backbone 2 and ilvD Gene fragment 2 was used to obtain ligation product 2.

[0103] Connecting linearized plasmid backbone 3 and ilvD Gene fragment 2 was used to obtain ligation product 3.

[0104] Connecting linearized plasmid backbone 3 and ilvD Gene fragment 3 was used to obtain ligation product 4.

[0105] Connecting linearized plasmid backbone 4 and ilvD Gene fragment 3 was used to obtain ligation product 5.

[0106] Based on the correspondence between the linearized plasmid backbone and the gene fragment described above, 3 μL of the gene fragment and 2 μL of the linearized plasmid backbone were taken and ligated using CE ligase in a PCR instrument at 50℃ for 15 min. The ligation products were then thoroughly mixed and transformed into 100 μL of DH5α-competent cells. The cells were incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, and then placed on ice for 2 min. 800 μL of LB medium was added, and the cells were incubated at 37℃ and 200 rpm for recovery. 100 μL of the recovery buffer was plated onto an Amp plate and incubated overnight at 37℃. Transformants were passaged on LB agar plates containing ampicillin for 1 h. Colony PCR was performed using primers AY3368 / AY3147. A 2200 bp band was considered a positive clone and sent to the company for sequencing. Recombinant plasmids with different linker lengths were successfully obtained and named Y398 (0 aa), Y396 (10 aa), Y415 (15 aa), Y394 (20 aa), and Y416 (25 aa), respectively. These five recombinant plasmids were transformed into Y315 competent cells to construct recombinant strains Y410 (0 aa), Y408 (10 aa), Y422 (15 aa), Y406 (20 aa), and Y423 (25 aa), as shown in Table 2.

[0107] Table 2 Recombinant strains with different linker lengths II. Effects of Linker Lengths on Isoleucine Yield To verify Bcd - ilvD The effect of linkers of different fusion protein lengths on isoleucine synthesis was investigated by fermenting recombinant strains Y410 (0 aa), Y408 (10 aa), Y422 (15 aa), Y406 (20 aa), and Y423 (25 aa), with strain Y424 (transformed into the control plasmid, i.e., the plasmid does not contain the bcd and ilvD genes) as the control. The specific fermentation methods are as follows: Each strain was fermented in shake flasks in MOPS medium containing 20 g / L glucose and 30 g / L threonine. After culturing at 37°C and 200 rpm for 36 h, samples were taken to determine cell density and isoleucine production.

[0108] Fermentation results as follows Figure 1 As shown in the figure. The results showed that the linker-free fusion (Y410) was not effective, with a yield of 7.20 g / L, even slightly lower than the control strain (7.41 g / L), and OD... 600The OD200 was 23.73, comparable to the control. The results indicate that direct fusion without a linker not only failed to increase yield but may have also led to functional impairment due to interference between the two enzymes, resulting in a slight decrease in yield. The 10 aa and 15 aa linkers brought slight increases, with both Y408 and Y422 yielding 7.89 g / L, approximately 6.5% higher than the control strain. 600 The OD values ​​were 26.41 and 24.54, respectively. This indicates that the spatial arrangement of the two enzymes was improved after the introduction of the linker, but the lengths of 10 aa and 15 aa were still insufficient to form an efficient metabolic pathway, resulting in limited improvement. The OD of Y408... 600 The results were higher than those of Y422, indicating that the 10 aa Linker had a superior growth-promoting effect. The 20 aa Linker achieved a breakthrough improvement, with Y406 showing an isoleucine yield as high as 12.99 g / L, a 75.3% increase compared to the control strain (7.41 g / L), and OD... 600 It reached 27.92, which is also the highest among all strains.

[0109] This result demonstrates that the 20 aa flexible linker (GGGGS)4 provides the optimal spatial distance for IlvD and Bcd, enabling both enzymes to maintain their correct folding while forming an efficient "metabolic pathway." The intermediate KMV produced by IlvD is efficiently captured and transformed by Bcd, greatly improving the overall kinetic efficiency of the continuous reaction.

[0110] The effect of 25 aa Linker declined, with Y423 yield at 7.52 g / L, only a 1.5% increase compared to the control, and OD 600 The result was 23.86, comparable to the control. This indicates that an excessively long linker makes the two enzymes spatially independent, allowing intermediate products to easily diffuse into the cytoplasm, weakening the channel effect, and causing the yield to drop back to baseline levels.

[0111] The linker length effect follows this pattern: from no linker to 20 aa, output increases with linker length, peaking at Y406; beyond 20 aa, output drops sharply. This trend exhibits a typical "bell curve," clearly indicating the existence of an optimal linker length window.

[0112] Overall analysis showed that 20 aa flexible Linker (GGGGS)4 was the optimal choice. The isoleucine production of the fusion protein strain Y406 reached 13.0 g / L, a 75.3% increase compared to the control strain. OD 600The yield was 27.92, setting a new record for shake-flask yield in this invention. Linker length has a decisive influence on the function of the fusion protein. From no linker to 20 aa, the yield increases with increasing linker length; the effect weakens after exceeding 20 aa, indicating an optimal length window. Linker-free fusion was ineffective; the yield of Y410 (7.2 g / L) was even lower than the control, suggesting that direct fusion may impair enzyme function. The 10 aa and 15 aa linkers showed limited improvement, with yields only increasing by 6.5% compared to the control, far less than the 75.3% increase of the 20 aa linker. The metabolic pathway effect was directly verified; the high yield of Y406 indicates that the 20 aa linker successfully achieved spatial proximity between IlvD and Bcd, enabling efficient transfer of intermediate products.

[0113] Example 4 I. Construction of Recombinant Strains with Different Ligation Sequences To verify the effect of fusion protein linker sequence on isoleucine synthesis, the optimal 20 aa flexible linker (GGGGS)4 determined in the previous step was used as the main research object. A 10 aa linker and no linker were also used as auxiliary validations to construct fusion proteins with different linker sequences. For forward fusion, IlvD was placed at the N-terminus and Bcd at the C-terminus, constructing three fusion genes: IlvD-20aa-Bcd, IlvD-10aa-Bcd, and IlvD-0aa-Bcd. For reverse fusion, Bcd was placed at the N-terminus and IlvD at the C-terminus, constructing three fusion genes: Bcd-20aa-IlvD, Bcd-10aa-IlvD, and Bcd-0aa-IlvD. All fusion genes were constructed using overlap extension PCR.

[0114] Using AY3369 / AY3370 as primers and MG1655 as a template, PCR was performed to obtain samples carrying RBS. ilvD Excerpt.

[0115] Using AY3344 / AY3370 as primers, and the above-mentioned ilvD Using a template, a PCR reaction was conducted to obtain a sample containing P. J23105 Homologous arms of 20 AA linkers / 10 AA linkers ilvD Segment 1.

[0116] Using AY3344 / AY3377 as primers, and the above-mentioned ilvD Using a template, a PCR reaction was conducted to obtain a sample containing P. J23105 Homologous arms with 0 aa linkers ilvD Segment 2.

[0117] Using AY3345 / AY3372 as primers and Y383 plasmid as template, a linearized vector backbone with 20 aalinker homologous arms was obtained by PCR reaction.

[0118] Using AY3345 / AY3374 as primers and Y383 plasmid as template, a linearized vector backbone with 10 aalinker homologous arms was obtained by PCR reaction.

[0119] Using AY3345 / AY3378 as primers and Y383 plasmid as template, a linearized vector backbone with 0 aalinker homologous arms was obtained by PCR reaction.

[0120] connect ilvD Fragment 1 and linearized carrier skeleton 1 are combined to obtain connection product 1.

[0121] connect ilvD Fragment 1 and linearized carrier skeleton 2 are combined to obtain linker product 2.

[0122] connect ilvD Fragment 2 and linearized carrier skeleton 3 are combined to obtain linker product 3.

[0123] The ligation products were obtained by ligating the fragments to the linearized vector backbone according to their correspondence. Specifically, 3 μL of the fragment and 2 μL of the linearized vector backbone were ligated using CE ligase in a PCR instrument at 50℃ for 15 min. After mixing each ligation product thoroughly, they were transformed into 100 μL of DH5α-competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, and then placed on ice for 2 min. 800 μL of LB medium was added, and the cells were incubated at 37℃ and 200 rpm for recovery. 100 μL of the recovery solution was plated onto an Amp plate and incubated overnight at 37℃. Transformants were passaged on LB plates containing ampicillin for 1 h. Colony PCR was performed using primers AY3343 / AY3375. A 2200 bp band was considered a positive clone and sent to the company for sequencing. Recombinant plasmids with different linker lengths were successfully obtained and named recombinant plasmids Y395, Y397, and Y399, respectively. The three recombinant plasmids were transformed into Y315 competent cells to construct recombinant strains Y407 (20aa), Y409 (10aa), and Y411 (0aa). The specific ligation order and linker length are shown in Table 3.

[0124] Table 3 Recombinant strains with different ligation sequences II. Effects of different connection sequences on isoleucine production Strains Y407, Y409, Y411, Y406, Y408, and Y410 were subjected to shake-flask fermentation in MOPS medium containing 20 g / L glucose and 30 g / L threonine, respectively. After incubation at 37°C and 200 rpm for 36 h, samples were taken to determine cell density and isoleucine production. The fermentation results are as follows: Figure 2 As shown.

[0125] Comparison of Linker-free fusion: Without the linker, the yield of Y411 (IlvD-Bcd) was 7.78 g / L, and the OD... 600 The yield was 26.32; the yield of Y410 (Bcd-IlvD) was 7.20 g / L, and the OD... 600 The value was 23.73. The yield and OD of Y411 were both higher than those of Y410, indicating that in the absence of a linker, the order of IlvD first and Bcd last is slightly better than the reverse order, but the difference is not significant and both are at a low level.

[0126] Comparison of 10aa Linker: The yield of Y408 (Bcd-10aa-IlvD) was 7.89 g / L, OD 600 The yield was 26.41; the yield of Y409 (IlvD-10aa-Bcd) was 7.49 g / L, OD 600 The value was 23.97. The yield and OD of Y408 were both higher than those of Y409, indicating that at a linker length of 10 aa, the order of Bcd first and IlvD last is better, but the difference between the two is still not significant.

[0127] Comparison of 20aa Linker: Y406 (Bcd-20aa-IlvD) yielded a high output of 12.94 g / L, OD 600 The concentration reached 27.92; while the yield of Y407 (IlvD-20aa-Bcd) was only 7.48 g / L, OD 600 The yield was 26.80. The yield of Y406 was 1.73 times that of Y407, a significant advantage. This result directly demonstrates that, at the optimal linker length, the linking sequence has a decisive influence on the function of the fusion protein.

[0128] Overall analysis showed that Bcd-20aa-IlvD (Y406) was the optimal fusion protein conformation, with an isoleucine yield of 12.94 g / L and OD... 600The yield was 27.92, an improvement of over 60% compared to other strains. The linker sequence has a decisive impact on fusion protein function; with a 20 aa linker, the Bcd-before-IlvD sequence (12.94 g / L) is significantly superior to the reverse sequence (7.48 g / L). Linker length and linker sequence exhibit a synergistic effect; the Bcd-before-IlvD sequence benefits from linker optimization (yield increased from 7.20 g / L to 13.0 g / L), while the reverse sequence cannot be improved through linker optimization. The metabolic pathway effect is directly verified; the high yield of Y406 demonstrates that the spatial organization of "upstream enzyme last, downstream enzyme first" aligns with metabolic flux, enabling efficient transfer of intermediate products.

[0129] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fusion protein for increasing isoleucine production, characterized in that, The fusion protein is connected to Bcd protein, Linker and IlvD protein sequentially from N-terminus to C-terminus; The amino acid sequence of the Bcd protein is shown in SEQ ID NO.1; The amino acid sequence of the IlvD protein is shown in SEQ ID NO.3; The Linker is (GGGGS) n Where n is 4.

2. A gene encoding the fusion protein as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

6.

4. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 2 or 3.

5. The use of the encoding gene as described in claim 2 or 3 or the recombinant expression vector as described in claim 4 in constructing a recombinant strain that produces high levels of isoleucine.

6. A method for constructing an engineered bacterium that produces isoleucine, characterized in that, The method includes the step of converting the encoding gene described in claim 2 or 3 into a chassis strain to construct a recombinant strain; the recombinant strain is the engineered bacterium.

7. The construction method according to claim 6, characterized in that, The strains in the chassis are Escherichia coli, Corynebacterium, or Bacillus.

8. The construction method according to claim 7, characterized in that, The Escherichia coli is Escherichia coli Y315; The *E. coli* Y315 strain was constructed from *E. coli* MG1655 by knocking out the ilvA, rhtA, tdcG, tdcE, ldhA, and brnQ genes, and overexpressing the ilvA*, ilvE, ilvIH*1, ilvIH*2, ilvC-ec, and ilvD genes. The nucleotide sequence of the ilvA* gene is shown in SEQ ID NO. 8; The nucleotide sequence of the ilvIH*1 gene is shown in SEQ ID NO.13; The nucleotide sequence of the ilvIH*2 gene is shown in SEQ ID NO.14; The nucleotide sequence of the ilvC-ec gene is shown in SEQ ID NO.

18.

9. An engineered bacterium constructed according to any one of claims 6-8.

10. A method for producing isoleucine by fermentation, characterized in that, The method includes the step of fermenting and culturing the engineered bacteria described in claim 9 to obtain isoleucine.

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