A high-activity malonyl-coa reductase mutant, and a modification method and application thereof

Abstract

The application discloses a high-activity malonyl-CoA reductase mutant and a modification method and application thereof, and belongs to the field of bioengineering. In view of the problem that the reverse reaction catalytic efficiency of a natural enzyme is low and restricts metabolic flux, a conformation gate site Glu234, a coenzyme loading site Pro221 and Thr232 are selected to carry out semi-rational site-directed mutation. Space steric hindrance is weakened through small-volume amino acid substitution, the combination adaptability of the oxidized coenzyme is optimized, and the conformation transformation energy consumption is reduced. Advantageous combination mutants such as E234S, E234Q, T232N single-point mutants and T232N-E234S are screened, and the specific enzyme activity of the optimal double mutant is 2.7 times that of the wild type. The mutant enzyme is applied to a non-carboxylated malonyl-CoA synthesis pathway, and the yield of the engineered bacteria for synthesizing aurovertin is increased by 3.3 times. The application effectively improves the precursor supply capacity and can be used for biosynthesis of polyketone, flavone and fatty acid natural products, and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a highly active malonyl-CoA reductase mutant, its modification method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Malonyl-CoA (M-CoA) is a core precursor in the biosynthesis of many important natural products, including polyketides, flavonoids, and fatty acids. It is derived from *Curvus orangeensis* (…). Chloroflexus aurantiacus Malonyl-CoA reductase (MCR) is a bifunctional enzyme that depends on NADPH. It performs aldehyde reduction and thioester reduction at the N-terminus and C-terminus, respectively. In the natural metabolic pathway, it mainly catalyzes the two-step reduction of M-CoA to 3-hydroxypropionic acid.

[0004] The C-terminus of the MCR (MCR-C) can reversibly catalyze the formation of M-CoA from malonate semialdehyde (MSA). The accumulation level of M-CoA within the bacterial cell is dynamically regulated by the downstream fatty acid synthesis capacity, and is very low. Previous studies have explored the introduction of a non-carboxylative malonyl-CoA pathway (NCM), specifically through *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa BauA and ) source Chloroflexus aurantiacus The MCR-C derived from pyruvate enables the production of M-CoA, increasing its intracellular level; however, the low catalytic efficiency of the reversible reaction of MCR-C has become a major bottleneck limiting metabolic flux.

[0005] The catalysis of MCR-C does not solely rely on the recognition of local active sites, but is a dynamic process tightly coupled with cap domain shift, hairpin ring ordering, and the formation of a catalytically closed state. MCR-C binds to the coenzyme in an open state, followed by substrate entry into the active site, inducing hairpin formation in the 226–238 region. This simultaneously drives cap domain closure, allowing the enzyme to enter a catalytically closed state, where chemical transformation is completed. The product is then released and the enzyme returns to the open state. During catalysis, Ser174 and Tyr192 form oxygen anion holes, while Tyr186, Arg189, Tyr192, and Arg228 participate in substrate / intermediate stabilization. Arg228 also plays a crucial gating role in hairpin ring conformational changes and cap closure. Existing structural and mutagenesis results indicate that the formation of an effective catalytic complex by MCR-C essentially depends on the synergistic matching of substrate / coenzyme loading, the catalytic center microenvironment, and conformational transitions. However, in the reverse reaction direction, MCR-C requires the catalytic exchange of MSA and NADP. + It assembles with CoA to form an efficient catalytic complex that favors the closed catalytic state; however, due to the natural evolutionary bias of this enzyme towards forward reduction reactions, there is a natural mismatch in its utilization of oxidized coenzymes, assembly of reverse reaction substrates, and induction of the closed state, thus significantly limiting its reverse reaction catalytic efficiency.

[0006] Therefore, this invention performs semi-rational modification of MCR-C based on catalytic mechanism from two levels: conformational gating and coenzyme loading, systematically improving the reverse reaction catalytic efficiency of MCR-C, thereby providing a key enzymatic basis for the efficient synthesis of M-CoA in the NCM pathway. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention provides the following technical solution: In a first aspect, a method for modifying malonyl-CoA reductase to enhance its reverse reaction catalytic ability is provided, the method comprising site-directed mutagenesis of the conformational gating site and / or coenzyme loading site of malonyl-CoA reductase.

[0008] Furthermore, the site-directed mutagenesis employs a gradient-style small-volume or weakened negative charge replacement, that is, replacing the amino acid at the mutation site with a smaller amino acid or replacing it with a decharged, weakened-occupancy amino acid type, thereby reducing the conformational cost for the enzyme to enter the closed peptide.

[0009] In one embodiment verified by the present invention, the MCR-C N395V K5561W S569R (MCR-C WT) reported in the prior art is used as the starting sequence (SEQ ID NO:1). The mutation site of the above conformational gating site is Glu234, and feasible mutation methods are such as: E234A, E234G, E234S, E234D, E234Q; the mutation sites of the coenzyme loading site are Pro221 and Thr232, and feasible mutation methods are such as: P221F, P221Y, T232D, T232N, T232S; this embodiment also includes combined mutations of the above mutation sites, further including any combination of two or three mutation sites.

[0010] As verified by this invention, in the above embodiments, the mutation methods with better effects are E234S, E234Q, T232N, or combined mutations T232N-E234Q and T232N-E234S; in the embodiment with the best effect, the T232N-E234S combined mutation results in the most significant improvement in the reverse reaction catalytic ability of malonyl-CoA reductase.

[0011] Secondly, a mutant of highly active malonyl-CoA reductase is provided, wherein the mutant is obtained by using the amino acid sequence shown in SEQ ID NO:1 as the starting sequence and undergoing E234S, E234Q, T232N or combined mutations T232N-E234Q, T232N-E234S.

[0012] The amino acid sequence of the T232N mutant is shown in SEQ ID NO: 2; The amino acid sequence of the E234Q mutant is shown in SEQ ID NO: 3; The amino acid sequence of the E234S mutant is shown in SEQ ID NO: 4; The amino acid sequence of the T232N-E234Q mutant is shown in SEQ ID NO: 5; The amino acid sequence of the T232N-E234S mutant is shown in SEQ ID NO: 6.

[0013] Thirdly, a gene sequence is provided that encodes the mutant described in the second aspect.

[0014] It should be noted that the gene sequence described in the third aspect includes any sequence that can be translated into the mutant described in the second aspect due to codon degeneracy, and the coding nucleic acid carrying the gene sequence is not limited to DNA or RNA; preferably, the coding nucleic acid is DNA, including cDNA, genomic DNA or artificially synthesized DNA; the DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0015] Fourthly, an expression cassette comprising the gene sequence described in the third aspect is provided.

[0016] Fifthly, a recombinant vector is provided, the recombinant vector comprising the complete coding reading frame sequence of the gene sequence described in the third aspect, or the recombinant vector comprising the expression cassette described in the fourth aspect.

[0017] The recombinant vector includes, but is not limited to, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors; further, the recombinant vector is a bacterial plasmid or a yeast plasmid. The construction method of the recombinant vector is conventional to those skilled in the art, for example, it can be constructed using in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. More specifically, it can be constructed by inserting the isolated polynucleotides into the multiple cloning site of the recombinant vector.

[0018] In a sixth aspect, an engineered bacterium is provided, wherein the engineered strain has a mutant of the malonyl-CoA reductase described in the second aspect.

[0019] Methods for modifying the starting strain include, but are not limited to, site-directed mutagenesis using overlap extension PCR (SOE-PCR), site-directed mutagenesis using QuikChange, site-directed saturation mutagenesis using SSM, multi-site synchronous mutagenesis, Gibson assembly, Golden Gate assembly, CRISPR-Cas-mediated site-directed editing, transposon mutagenesis, and codon optimization.

[0020] In other embodiments verified by this invention, the engineered bacteria, compared to the starting strain, also possess an enhanced NCM expression pathway; further, the enhanced NCM expression pathway is achieved by introducing BauA and RppA genes or increasing their copy number. In the embodiments verified by this invention, the amino acid sequence of BauA is shown in SEQ ID NO: 7, and the amino acid sequence of RppA is shown in SEQ ID NO: 8. The modification method provided by this invention targets malonyl-CoA reductase in microorganisms, therefore this modification method can be widely applied. The above-mentioned mutation method is aimed at modifying malonyl-CoA reductase in microorganisms. The present invention further provides a method for introducing NCM expression pathway into microorganisms. Therefore, the starting strain of the engineered bacteria can be common industrial bacteria, fungi or archaea.

[0021] Furthermore, viable bacteria include, but are not limited to, Escherichia coli (such as Escherichia coli). Escherichia coli ), Pseudomonas (such as Pseudomonas putida) Pseudomonas putida Paracoccus (such as denitrifying paracoccus) Paracoccus denitrificans ), genus *Columbia* (such as *Columbia orangeensis*) Chloroflexus aurantiacus ), genus Curvature of Rose (such as Curvature of Rose) Roseiflexus castenholzii ), Bacillus species (such as Bacillus subtilis) Bacillus subtilis Corynebacterium spp. (such as Corynebacterium glutamicum) Corynebacterium glutamicum Lactobacillus (such as Lactobacillus plantarum) Lactiplantibacillus plantarum Lactobacillus acidophilus Lactobacillus acidophilus Streptomyces (such as Streptomyces azureense) Streptomyces coelicolor Streptomyces griseus Streptomyces griseus Streptomyces limonene Streptomyces lividans One of them.

[0022] Furthermore, feasible fungi include, but are not limited to, yeasts (such as Saccharomyces cerevisiae). Saccharomyces cerevisiae Pichia genus (such as Pichia pastoris) Komagataella pastoris Yersinia genus (such as Yersinia lipolytica) Yarrowia lipolytica Aspergillus species (such as Aspergillus niger) Aspergillus niger Aspergillus oryzae Aspergillus oryzae ), Trichoderma (such as Trichoderma reesei) Trichoderma reesei One of them.

[0023] Furthermore, feasible archaea include, but are not limited to, the genus *Metallococci* (such as *Metallococci*). Metallosphaera sedula ), Sulfidophytes (such as acidophilic thermosulfidophytes) Sulfolobus solfataricus East Asian sulfur leaf fungus Sulfolobus tokodaii One of them.

[0024] In a seventh aspect, the application of a mutant of the malonyl-CoA reductase described in the second aspect or the engineered bacteria described in the sixth aspect is provided, the application including at least the synthesis of polyketide compounds, flavonoid compounds or fatty acid components.

[0025] The flavonoids include, but are not limited to, erythromycin, tetracycline, triacetin lactone, phloroglucinol, naringenin, dihydroquercetin, resveratrol, curcumin, and derivatives of the above compounds; in one embodiment verified by the present invention, the mutant is transformed into Escherichia coli for the synthesis of aflatoxin.

[0026] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is based on the mechanism of enzyme catalytic conformational change and coenzyme binding. It employs semi-rational site-directed mutagenesis targeting conformational gating sites and key coenzyme loading sites to effectively reduce the energy barrier to enzyme conformational closure, optimize the adaptability of oxidized coenzyme loading, and significantly enhance the reverse reaction catalytic activity of malonyl-CoA reductase. The single mutant enzyme activity is up to 74% higher than the wild type, and the dual-site combined mutant T232N-E234S achieves a catalytic specific enzyme activity up to 2.7 times that of the wild type, significantly breaking through the original catalytic efficiency bottleneck.

[0027] 2. The highly active mutant enzyme obtained through modification can significantly enhance the metabolic flux of the non-carboxylated malonyl-CoA synthesis pathway and effectively increase the intracellular supply of malonyl-CoA precursors. After constructing engineered strains using this mutant enzyme, the yield of aflatoxin synthesis increased by 3.3 times compared to the wild system, providing sufficient precursor support for the biosynthesis of polyketides, flavonoids, and fatty acid natural products.

[0028] 3. This invention clarifies the role of key sites such as E234 and T232 in enzyme conformation regulation and coenzyme recognition. The mutation modification method is simple and controllable, the mutant gene is easy to construct recombinant vectors and stably expressed in common engineered microorganisms, and the strain culture and catalytic application conditions are mild, showing good prospects for industrial biosynthesis applications. The obtained mutants and recombinant engineered bacteria are highly versatile and can be adapted to a variety of chassis microorganisms, enabling them to be widely used in the creation of various natural products using malonyl-CoA as a synthetic precursor, expanding the types of microbial synthetic products and production efficiency. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 This is a diagram showing the interaction between the E234 residue in the open state and surrounding residues as described in the Specific Implementation section.

[0031] Figure 2 The cofactor NADP described in the specific implementation section + Diagram showing the relative positions of the nicotinamide ring and surrounding residues P221 and T232; in, Figure 2 A is in an open state. Figure 2B is in a closed state.

[0032] Figure 3 The results show the relative enzyme activity of the crude enzyme solution of the single-point mutant described in Example 1; in, Figure 3 The relative enzyme activity of the crude enzyme solution is as follows: A represents a single mutation at the E234 site. Figure 3 The relative enzyme activity of crude enzyme solution with single mutation at sites P221 and T232 is determined by B.

[0033] Figure 4 The results are for the pure enzyme activity described in Example 1; in, Figure 4 In the A group, the dominant mutant at the E234 site showed higher enzyme activity than the pure enzyme. Figure 4 The enzyme activity of the pure enzyme in the B-type mutant with the T232N site was higher than that of the enzyme in the T232N site.

[0034] Figure 5 The results are for the pure enzyme activity of the combined mutant described in Example 1.

[0035] Figure 6 The results show the THN levels synthesized from the wild-type MCR-C and the combined mutant T232N E234S described in Example 2. Detailed Implementation

[0036] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0038] In the context of this invention, the word "comprising" is considered to mean "particularly including". It should not be interpreted as "consisting of only".

[0039] In the description of this invention, it should be understood that the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. The symbol " / " in this document indicates that the related objects are in an "or" relationship; for example, A / B means A or B.

[0040] In the description of embodiments of the present invention, the words "exemplary" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of the present invention should not be construed as being more preferred or advantageous than other embodiments or design. Specifically, the use of the words "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0041] In the description of the embodiments of the present invention, unless otherwise stated, "a plurality of" means two or more.

[0042] As described in the background section, the existing MCR-C reversible reaction has low catalytic efficiency, which has become a major bottleneck limiting metabolic flux. In order to solve the above technical problems, this invention designs a method to obtain higher catalytic efficiency through targeted mutation.

[0043] Based on the catalytic principle of MCR-C, this invention aims to improve the reverse reaction catalytic performance by carrying out multi-level semi-rational modification of MCR-C around the key links that limit the efficiency of the reverse reaction. The main schemes include: conformational gating site modification and coenzyme loading site modification.

[0044] This invention uses the previously reported MCR-C N395V K5561W S569R (MCR-C WT) as the initial sequence and modifies it, the amino acid sequence of which is shown in SEQ ID NO:1.

[0045] 1. Conformation-gated site modification like Figure 1 As shown, in the open state, the Glu234 residues occupy the binding site of the malonyl group in M-CoA. Substrate entry into the binding site requires a certain degree of conformational change in Glu234 to provide sufficient space. In the forward reaction, MCR-C has a strong affinity for M-CoA, and the larger size of M-CoA allows it to break the relevant intermolecular hydrogen bonds and enter the substrate binding site. However, in the reverse reaction, the free MSA molecule is smaller and has a weaker interaction with the enzyme. The free energy released during its binding is insufficient to break the intramolecular hydrogen bonds formed by Glu234, thus preventing it from entering the binding site and driving the conformational transition of MCR-C from the open state to the catalytically closed state. To facilitate MSA entry into the binding site and initiation of the reverse reaction, this invention performs site-directed mutagenesis on the conformational gating site Glu234, employing a gradient of small-volume or weakened negative charge substitutions. By mutating to small-volume Ala, Gly, or decharged / weakened occupancy Ser, Gln, Asp, the conformational cost of the enzyme transitioning from the open state to the catalytically relevant closed state is reduced. The catalytic efficiency of each mutant is evaluated, and the optimal mutant is selected.

[0046] 2. Modification of coenzyme loading sites MCR-C naturally catalyzes reduction reactions, therefore its active site is better suited for NADPH-driven forward catalysis; while in the reverse reaction, the cofactor is NADP. + It assembles with MSA and CoA to form an efficient catalytic complex that favors the formation of a closed catalytic state. Existing structural results indicate that the coenzyme binding of MCR-C is not a static process, but is tightly coupled with the conformational transition from open to closed: Tyr192 / Lys196 / Arg228 constitute a key anchoring network for NADP, while Thr232 is close to the coenzyme in the open state and far away in the closed state; simultaneously, the local interaction pattern around the nicotinamide ring also changes after the cap closes. That is, NADP... + The correct loading is closely related to the efficiency of the reverse reaction.

[0047] NADP + The interaction between the nicotinamide ring and surrounding residues, such as Figure 2 As shown: A represents the open state, and B represents the closed state. Pro221 is located on the periphery of the nicotinamide ring and forms an alkyl-π interaction with the nicotinamide ring, which is a relatively weak hydrophobic interaction. NADP + The nicotinamide ring in the oxidized state is close to a planar conformation, making it more susceptible to benefiting from aromatic / hydrophobic clamping. Therefore, Pro221 was mutated to Phe / Tyr containing an aromatic ring, thereby improving NADP by altering the aromatic constraint near the nicotinamide ring. + The loading of Thr232 showed that it only had a short-range interaction with the nicotinamide ring in the open state, while in the closed state, this interaction disappeared as the hairpin structure moved away from it. Therefore, Thr232 was mutated to Asn / Ser to optimize NADP. + The transition process from initial loading to the closed state, through the above modifications, aims to improve NADP. + Mutants loaded and effectively utilized in the reverse reaction direction.

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0049] Example 1 (I) Construction of MCR-C mutant The MCR-C WT gene was synthesized and cloned into the pET28a(+) plasmid. Nco I and HinThe dIII restriction site was modified, and a 6×His Tag was introduced at its C-terminus. This was accomplished by Beijing Qingke Biotechnology Co., Ltd., resulting in the pET28a-MCR-C recombinant plasmid. Using the pET28a-MCR-C plasmid as a template, MCR-C gate site E234 mutants (E234A, E234G, E234S, E234D, E234Q) and coenzyme loading site P221 and T232 mutants (P221F, P221Y, T232D, T232N, T232S) were constructed via whole-plasmid PCR. Primers are shown in Table 1. The PCR products were digested with DpnI digestive enzyme for 1 h, followed by DNA purification and transformation to... Escherichia coli Gene sequencing was performed on DH5α positive transformants.

[0050] Table 1 Primers used for constructing mutant plasmids (II) Expression and enzyme activity detection of wild-type enzyme WT and various mutants (1) Expression of recombinant strains The WT expression vector and the correctly sequenced mutant expression vectors from Example 1 were transformed into... E. coli BL21(DE3) competent cells. Single clones were picked and activated to obtain seed culture. The seed culture was then mixed with 1% ( v / v Transfer to a 250 mL Erlenmeyer flask containing 100 mL LB medium (kanamycin final concentration 50 μg / mL), and incubate at 37°C with shaking at 200 rpm until OD. 600 When the concentration is 0.8-1.0, add IPTG to a final concentration of 0.2 mM and induce at 25℃ for 12-16 h. After induction, collect the bacterial cells by centrifugation.

[0051] (2) Screening of enzyme activity in crude enzyme solution The collected bacterial cells were reconstituted with Tris buffer (pH 7.8) at a concentration of 10%, and then sonicated. The supernatant was collected, filtered through a 0.45 μm filter membrane, and used as the crude enzyme solution for in vitro enzyme activity screening. The total reaction volume was 200 μL, containing 50 mM Tris-HCl (pH 7.8), 1.5 mM EDTA, 1 mM MgCl2, 2.5 mM pyruvate, 0.5 mM β-alanine, and 2.5 mM NADP. +2.5 mM CoA, 0.45 μM BauA protein, and 20-fold diluted MCR-C crude enzyme solution were added. After adding all reagents except CoA, the mixture was incubated at room temperature for 3-5 min, and finally CoA was added. The absorbance at 340 nm was measured using a microplate reader in kinetic mode (to monitor NADPH production) for a total of 5 min, with measurements taken every 15 s. The crude enzyme concentration was simultaneously measured using Bradford assay to ensure similar protein concentrations in the crude enzyme solution. The relative enzyme activities of WT and each mutant are as follows: Figure 3 As shown, the relative enzyme activities of the gate site mutants E234S and E234Q were 172% and 126%, respectively, and the relative enzyme activity of the coenzyme loading site mutant T232N was 145%, all of which were significantly higher than those of WT.

[0052] (3) Protein purification and enzyme activity assay The crude enzyme solution was purified by affinity chromatography (AKTA system equipped with 5 mL HisTrap HP): The remaining crude enzyme solution was loaded at a flow rate of 5 mL / min, followed by washing with binding buffer (50 mM Tris-HCl, 200 mM NaCl, pH 8.0) until all unbound protein was eluted. Four column volumes of contaminating protein were eluted using a buffer of 50 mM Tris-HCl, 200 mM NaCl, and 50 mM imidazole at pH 8.0. Finally, the target protein was eluted using an elution buffer of 50 mM Tris-HCl, 200 mM NaCl, and 300 mM imidazole at pH 8.0. Protein concentration was quantified using A280 and extinction coefficient. SDS-PAGE analysis showed that all proteins exhibited single bands, indicating high purification quality. The purified protein was concentrated using a 10 kDa ultrafiltration tube, and the buffer was replaced with 50 mM Tris-HCl (pH 7.8).

[0053] Enzyme activity was detected using purified enzyme, with the reaction system as described above. MCR-C was performed using 0.15 μM purified enzyme. The specific enzyme activity results for the purified enzyme are as follows: Figure 4 As shown, in the modification of the gating site, the specific enzyme activity of WT was 121.8±14.14 U / mg, that of E234S was 211.8±3.86 U / mg, and that of E234Q was 154.5±2.57 U / mg, representing increases of 74% and 27% respectively compared to WT. In the modification of the coenzyme loading site, the specific enzyme activity of WT was 117.3±2.57 U / mg, and that of T232N was 172.7±5.14 U / mg, representing an increase of 47% compared to WT.

[0054] (III) Construction, expression and enzyme activity detection of combinatorial mutants (1) Construction of combinatorial mutants The results from "(II) Expression and Enzyme Activity Detection of Wild-type Enzyme WT and Various Mutants" show that the E234S, E234Q, and T232N mutations significantly increased the enzyme activity of the MCR-C reverse reaction. Therefore, two mutations of E234 were combined with T232N for mutation. Using the pET28a-MCR-C T232N plasmid as a template, whole-plasmid PCR was performed, with primers shown in Table 2. The PCR product was digested with DpnI digestive enzyme for 1 h, followed by DNA purification and transformation to... E. coli Gene sequencing was performed on DH5α positive transformants.

[0055] Table 2 Primers used for constructing combined mutant plasmids (2) Expression of the combined mutant strain The correctly sequenced expression vector was transformed into BL21(DE3) competent cells. Single clones were picked and activated to obtain seed culture. The seed culture was then diluted with 1% ( v / v Transfer to a 250 mL Erlenmeyer flask containing 100 mL LB medium (kanamycin final concentration 50 μg / mL), and incubate at 37°C with shaking at 200 rpm until OD. 600 When the concentration is 0.8-1.0, add IPTG to a final concentration of 0.2 mM and induce at 25℃ for 12-16 h. After induction, collect the bacterial cells by centrifugation.

[0056] (3) Protein purification and enzyme activity assay The combined mutant protein was purified, and its activity was detected using the purified enzyme. The total reaction volume was 200 μL, containing 50 mM Tris-HCl (pH 7.8), 1.5 mM EDTA, 1 mM MgCl2, 2.5 mM pyruvate, 0.5 mM β-alanine, and 2.5 mM NADP. + The enzyme contained 2.5 mM CoA, 0.45 μM BauA protein, and 0.15 μM MCR-C mutant protein. After adding all reagents except CoA, the enzyme was incubated at room temperature for 3–5 min. Finally, CoA was added, and the absorbance at 340 nm was measured using a microplate reader in kinetic mode (to monitor NADPH production). The total incubation time was 5 min, with measurements taken every 15 s. The specific enzyme activity of the purified enzyme is shown below. Figure 5 As shown, the specific enzyme activity of WT was 119.1±3.86 U / mg, that of T232N-E234Q was 226.4±3.86 U / mg, and that of T232N-E234S was 318.6±7.07 U / mg. T232N-E234S had the best enzyme activity, which was 2.7 times that of WT.

[0057] Example 2: Detection of aflatoxin synthesis level in mutants The improved catalytic efficiency of the MCR-C reverse reaction helps to increase the level of M-CoA synthesis via the NCM pathway, providing more precursors for the microbial synthesis of natural products using M-CoA as a precursor. 1,3,6,8-Tetrahydroxynaphthalene (THN) is a unique symmetrical polyketide-derived compound, produced directly from M-CoA and acetyl-CoA by polyketide synthases. It spontaneously forms the red compound aflatoxin, exhibiting distinct absorption peaks at 340 nm and 520 nm. Multiple studies have used... Streptomyces griseus The RppA protein from this source is used as an M-CoA sensor. This invention investigates the effect of increased MCR-C reverse reaction catalytic efficiency on the synthesis of natural products using M-CoA as a precursor in *E. coli* by co-expressing RppA in the NCM pathway.

[0058] (1) Construction of expression carrier: synthesis rppA and bauA The genes were cloned into the NcoⅠ and HindⅢ restriction sites of pETDuet-1 and pCDFDuet-1, respectively, by Beijing Qingke Biotechnology Co., Ltd., to obtain the recombinant plasmids pETDuet-RppA and pCDFDuet-BauA. Wild-type MCR-C and the optimal mutant MCR-C T232N-E234S obtained in Example 3 were constructed into the pCDFDuet-BauA vector to obtain the NCM pathway expression vectors pCDFDuet-BauA-MCR-C WT and pCDFDuet-BauA-MCR-C T232N-E234S.

[0059] (2) Expression and detection of aflatoxin: The NCM pathway expression vector and the aflatoxin expression vector pETDuet-RppA were simultaneously transformed into *E. coli* BL21(DE3). Three clones from each strain were selected for activation to obtain seed culture. The seed culture was then diluted with 1% (… v / v The cells were transferred to test tubes containing 3 mL of M9 medium (final concentration of streptomycin 50 μg / mL, final concentration of ampicillin 100 μg / mL, final concentration of IPTG 0.2 mM) and incubated at 37°C with shaking at 200 rpm for 24 h. After incubation, the OD of the bacterial culture was measured. 600 Simultaneously, 1 mL of bacterial culture was taken, centrifuged, and the supernatant was collected. The absorbance at 340 nm was measured using an ELISA reader, and the values ​​of A and B were compared. 340 / OD 600 The result is as follows Figure 6As shown, the modified NCM pathway increased the level of aflatoxin synthesis by 3.3 times compared to the wild type. This means that the modified MCR-C enables the NCM pathway to produce more malonyl-CoA, which helps to improve the biosynthetic capacity of microorganisms to synthesize natural products such as polyketides and flavonoids that use malonyl-CoA as a precursor.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for modifying malonyl-CoA reductase to enhance its reverse reaction catalytic ability, characterized in that, The modification method includes site-directed mutagenesis of the conformational gating site of malonyl-CoA reductase, or combined mutagenesis of the conformational gating site and the coenzyme loading site. The starting sequence of the malonyl-CoA reductase is shown in SEQ ID NO:1; The mutation site of the conformational gating site is Glu234, and the mutation mode is E234S, E234Q; The mutation site of the coenzyme loading site is Thr232, the mutation mode is T232N, and the combined mutations are T232N-E234Q and T232N-E234S.

2. A mutant of highly active malonyl-CoA reductase, characterized in that, The mutant was obtained by using the amino acid sequence shown in SEQ ID NO:1 as the starting sequence and undergoing E234S, E234Q or combined mutations T232N-E234Q and T232N-E234S. The amino acid sequence of the E234Q mutant is shown in SEQ ID NO: 3; The amino acid sequence of the E234S mutant is shown in SEQ ID NO: 4; The amino acid sequence of the T232N-E234Q mutant is shown in SEQ ID NO: 5; The amino acid sequence of the T232N-E234S mutant is shown in SEQ ID NO:

6.

3. A gene sequence, characterized in that, The gene sequence is used to encode the mutant of claim 2; the gene sequence includes any sequence that can be translated into the mutant of claim 2 due to codon degeneracy, and the coding nucleic acid carrying the gene sequence includes DNA or RNA.

4. An expression cassette comprising the gene sequence of claim 3.

5. A recombinant vector, characterized in that, The recombinant vector comprises the complete coding reading frame sequence of the gene sequence of claim 3, or the recombinant vector comprises the expression cassette of claim 4; The recombinant vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors.

6. An engineered bacterium, characterized in that, The starting strain of the engineered bacteria is Escherichia coli, and the engineered bacteria contains a mutant of malonyl-CoA reductase as described in claim 2; The method for modifying the starting strain is the QuikChange site-directed mutagenesis method; Compared to the original strain, the engineered bacteria also have an enhanced NCM expression pathway; the enhanced NCM expression pathway is achieved by introducing BauA and RppA encoding genes or increasing their copy number, the amino acid sequence of BauA is shown in SEQ ID NO: 7, and the amino acid sequence of RppA is shown in SEQ ID NO:

8.

7. The application of the mutant of malonyl-CoA reductase according to claim 2 or the engineered bacteria according to claim 6, characterized in that, The application is for the synthesis of lutein.

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