5-carboxyl-2-pentenoyl-coenzyme A reductase mutant and application thereof

Abstract

The invention relates to a 5-carboxyl-2-pentenoyl-coenzyme A reductase mutant and application thereof, and belongs to the technical field of biological engineering. Aiming at the problem of low cofactor binding efficiency of 5-carboxyl-2-pentenoyl-coenzyme A reductase in a reverse adipic acid degradation pathway, rational prediction and site-directed mutagenesis experiments are carried out by taking a gene for expressing the 5-carboxyl-2-pentenoyl-coenzyme A reductase as a research object, structural optimization is carried out on a binding site of the enzyme, and the cofactor binding efficiency of the 5-carboxyl-2-pentenoyl-coenzyme A reductase in the reverse adipic acid degradation pathway is improved. According to the method disclosed by the invention, the adipic acid is more suitable for the characteristics of cofactors, so that the binding capacity of the enzyme to the cofactors is enhanced, the activity of catalyzing the reaction of 5-carboxyl-2-pentenoyl-coenzyme A to generate adipoyl coenzyme A is improved, and the yield of the adipic acid is further improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a 5-carboxy-2-pentenoyl-CoA reductase mutant and its application. Background Technology

[0002] Adipic acid, also known as fatty acid, is an important platform compound. It is not only a core monomer for the production of nylon 66 and polyurethane, but is also widely used in the preparation of engineering plastics, plasticizers, high-grade lubricants, food additives, pharmaceutical intermediates and other diversified products.

[0003] However, the current industrial production of adipic acid still mainly relies on highly polluting and energy-intensive petrochemical routes. For example, the industrial production route primarily involves oxidizing a mixture of cyclohexanol and cyclohexanone with nitric acid. This process releases the greenhouse gases nitrogen dioxide and nitrous oxide, which have a strong greenhouse effect, and generates large amounts of highly concentrated acidic wastewater. Furthermore, this process faces safety and equipment corrosion issues and depends on petroleum-based feedstocks. Therefore, there is an urgent need to find a method to replace the traditional chemical synthesis of adipic acid.

[0004] With the development of biotechnology, the total biosynthesis method for producing organic acids is increasingly favored due to its advantages such as low production cost, simple production process, low pollution, and high product purity. In the biosynthesis of adipic acid, the reverse adipic acid degradation pathway is a major research focus. For example, CN108004275A discloses a recombinant Escherichia coli strain that produces adipic acid. Using E. coli BL21(DE3) as the host, it overexpresses the β-ketothiolase gene, 3-hydroxyacyl-CoA dehydrogenase gene, 3-hydroxyadipyl dehydrogenase gene, 5-carboxy-2-pentenoyl-CoA reductase gene, and adipyl-CoA in modules, replacing the promoter. The rate-limiting enzyme (5-carboxy-2-pentenoyl-CoA reductase) exhibits very low activity in catalyzing the reaction of 5-carboxy-2-pentenoyl-CoA to adipyl-CoA, thus affecting the efficiency of adipic acid synthesis.

[0005] In conclusion, the development of 5-carboxy-2-pentenoyl-CoA reductase with high catalytic activity is of great significance to the field of adipic acid biosynthesis. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a 5-carboxy-2-pentenoyl-CoA reductase mutant and its application. The invention rationally modifies 5-carboxy-2-pentenoyl-CoA reductase to improve its cofactor binding efficiency, aiming to achieve efficient synthesis of adipic acid.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a 5-carboxy-2-pentenoyl-CoA reductase mutant, wherein the amino acid sequence of the mutant is mutated based on the sequence shown in SEQ ID NO.2, and the mutated site includes R247 (arginine at position 247).

[0009] This invention addresses the problem of low cofactor binding efficiency of 5-carboxy-2-pentenoyl-CoA reductase in the reverse adipic acid degradation pathway. Using the gene expressing 5-carboxy-2-pentenoyl-CoA reductase as the research object, rational prediction and site-directed mutagenesis experiments were conducted to optimize the enzyme's binding site structure, making it more adaptable to the characteristics of cofactors. This enhances the enzyme's cofactor binding ability, improves its catalytic activity in the reaction of 5-carboxy-2-pentenoyl-CoA to adipicyl-CoA, and ultimately increases the yield of the target product, adipic acid.

[0010] Preferably, the mutation includes at least one of R247S (arginine at position 247 is mutated to serine), R247N (arginine at position 247 is mutated to asparagine), R247E (arginine at position 247 is mutated to glutamic acid), or R247A (arginine at position 247 is mutated to alanine).

[0011] SEQ ID NO.2:

[0012] MSDFDLYRPTEEHEALREAIRSVAEDKIAPHAADVDEQSRFPQEAYEALRASDFHAPHVAEEYGGVGADALATCIVIEEIARVCASSSLIPAVNKLGSMPLILSGSDEVKQRYLPELASGEAMFSYGLSEREAGSDTASMRTRAVRDGDDWILNGQKSWITNAGISKYYTVMAVTDPDGPRGRNISAFVVHID DPGFSFGEPERKLGIKGSPTRELIFDNVRIPGDRLVGKVGEGLRTALRTLDHTRVTIGAQAVGIAQGALDYALGYVKERKQFGKAIADFQGIQFML ADMAMKLEAARQMVYVAAAKSERDDDADLSFYGAAAKCFASDVAMEITTDAVQLLGGYGYTRDYPVERMMRDAKITQIYEGTNQIQRVVMARQLLKK.

[0013] It is understood that enzymes with similar functions obtained by modifying the 5-carboxy-2-pentenoyl-CoA reductase mutant designed in this invention through amino acid substitution, deletion, or addition using genetic modification methods in the art should be within the scope of protection of this invention. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence identity between the modified amino acid sequence and its corresponding original sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. Furthermore, enzymes obtained by conservatively substituting amino acids with similar or comparable properties are also within the scope of protection of this invention.

[0014] In a second aspect, the present invention provides a nucleic acid molecule that encodes the 5-carboxy-2-pentenoyl-CoA reductase mutant described in the first aspect.

[0015] In this invention, due to the degeneracy of the genetic code, a large number of nucleic acid molecules that can be used to encode the 5-carboxy-2-pentenoyl-CoA reductase mutant can be obtained. Therefore, given the identification of a specific amino acid sequence, those skilled in the art can easily prepare any number of different nucleic acids by changing the sequence of one or more codons without altering the amino acid sequence encoding the protein. Based on the preferences of the host cell used in the actual preparation process, codon optimization can be performed to select more preferred polynucleotides. The nucleic acid molecules can be obtained using conventional methods, such as PCR amplification or artificial synthesis.

[0016] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0017] Preferably, the starting vector of the recombinant vector includes a plasmid vector.

[0018] Preferably, the plasmid vector includes any one of the pRSFDuet series vectors, pET series vectors, pETduet series vectors, pREST series vectors, pETDuet series vectors, or pCDuet series vectors.

[0019] Fourthly, the present invention provides a recombinant bacterium containing the nucleic acid molecule described in the second aspect.

[0020] Preferably, the starting strain of the recombinant bacteria includes Escherichia coli and / or yeast.

[0021] Preferably, the Escherichia coli includes any one of the Escherichia coli K-12 series strains or the Escherichia coli B series strains.

[0022] Preferably, the yeast includes any one of Saccharomyces cerevisiae, Isaac's orientalis, or Pichia pastoris.

[0023] The recombinant bacteria of the present invention express the 5-carboxy-2-pentenoyl-CoA reductase mutant described in the first aspect, wherein the genome integrates a nucleic acid molecule encoding the 5-carboxy-2-pentenoyl-CoA reductase mutant or a recombinant vector containing said nucleic acid molecule.

[0024] Preferably, the recombinant bacteria also express β-ketothiolytic enzyme gene, 3-hydroxyacyl-CoA dehydrogenase gene, 3-hydroxyadiacyl-CoA dehydrogenase gene, and adipicyl-CoA synthase gene.

[0025] In this invention, genes related to the adipic acid synthesis pathway can be further expressed in recombinant bacteria, enabling efficient synthesis of adipic acid directly using recombinant bacteria.

[0026] Fifthly, the present invention provides a method for constructing the recombinant bacteria described in the fourth aspect, the method comprising:

[0027] The nucleic acid molecule described in the second aspect is inserted into the genome of the starting strain; or the recombinant vector described in the third aspect is introduced into the starting strain.

[0028] Based on the gene modification strategy designed in this invention, recombinant bacteria can be obtained using gene modification methods commonly used in the art. For example, the transformation of the vector into host cells can be carried out using conventional methods well known to those skilled in the art. These methods include the CaCl2 method, electroporation, and calcium phosphate co-precipitation, as well as conventional mechanical methods such as microinjection, electroporation, and liposome packaging. The obtained recombinant bacteria can be cultured using conventional methods well known to those skilled in the art. The culture medium can be a conventional medium. The 5-carboxy-2-pentenoyl-CoA reductase mutant produced by the recombinant bacteria can be separated and purified using physical and chemical methods, such as salting out, centrifugation, cell disruption, and chromatography.

[0029] In a sixth aspect, the present invention provides the application of the 5-carboxy-2-pentenoyl-CoA reductase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the recombinant bacteria described in the fourth aspect in the synthesis of adipic acid.

[0030] In a seventh aspect, the present invention provides a method for synthesizing adipic acid, the method comprising: utilizing the 5-carboxy-2-pentenoyl-CoA reductase mutant described in the first aspect to participate in the catalytic synthesis reaction of adipic acid.

[0031] This invention designs and modifies 5-carboxy-2-pentenoyl-CoA reductase to improve its cofactor binding ability and catalytic activity in the reaction of 5-carboxy-2-pentenoyl-CoA to produce adipicoyl-CoA. It can be effectively applied to catalyze the synthesis of adipic acid (reverse adipic acid degradation pathway). It can be synthesized in vitro by enzyme catalysis or by constructing a synthetic pathway within a bacterial strain to culture the corresponding recombinant bacteria to prepare adipic acid.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] This invention utilizes computational simulations to design mutants targeting amino acid residues near the binding site of 5-carboxy-2-pentenoyl-CoA reductase. These mutants are then screened based on adipic acid yield to obtain mutants that increase adipic acid production. Using these mutants can improve adipic acid yield, reduce production costs, and promote the industrial application of adipic acid. Attached Figure Description

[0034] Figure 1 This is a graph showing the energy changes after 5-carboxy-2-pentenoyl-CoA reductase binds to alanine around the 5 Å pocket.

[0035] Figure 2 Gel electrophoresis image of the mutant plasmid.

[0036] Figure 3A This is a chromatogram of the high-performance liquid chromatography (HPLC) results for the fermentation sample.

[0037] Figure 3B The image shows the high-performance liquid chromatography (HPLC) results for adipic acid standards.

[0038] Figure 4 The graph shows the adipic acid yield of recombinant bacteria containing different 5-carboxy-2-pentenoyl-CoA mutants. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0041] Example 1

[0042] This embodiment describes the molecular modification design of 5-carboxy-2-pentenoyl-CoA reductase.

[0043] Using the natural 5-carboxy-2-pentenoyl-CoA reductase gene (SEQ ID NO.1) as a template, the binding pocket of the protein was first predicted using COACH. Then, molecular docking was performed using Discovery Studio software to identify key amino acid residues within a 5 Å range of the ligand. Specific amino acid residues were replaced with residues capable of forming stronger hydrogen bonds or electrostatic interactions with the cofactor to enhance its binding ability. Alanine scan of the amino acids within 5 Å of the binding site revealed that site-directed mutagenesis resulted in the lowest docking energy value at position 247. Figure 1 Therefore, virtual saturation mutations were performed on it, and finally four mutations with the lowest binding energy changes were selected for subsequent experimental verification, namely R247S, R247N, R247E and R247A.

[0044] SEQ ID NO.1:

[0045]

[0046] Example 2

[0047] This embodiment describes the construction of a recombinant bacterium containing a mutant of 5-carboxy-2-pentenoyl-CoA reductase.

[0048] Primers were designed for the mutation sites R247S, R247N, R247E and R247A designed in Example 1 (Table 1). Site-directed PCR amplification was performed using the 5-carboxy-2-pentenoyl-CoA reductase gene as a template. The mutation sites were constructed by introducing site-directed mutagenesis using whole plasmid PCR.

[0049] Table 1

[0050]

[0051] The underlined sites in the table represent mutation sites.

[0052] The PCR reaction system consisted of: 2×PhantaMax Buffer (Vazyme), 1 μL of forward primer (10 μmol·L⁻¹). -1 ), 1 μL reverse primer (10 μmol·L -1 ), 1 μL template DNA, add distilled water to 50 μL.

[0053] The PCR amplification program was as follows: First, pre-denaturation at 98℃ for 4 min; then 30 cycles: denaturation at 98℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 8 min; finally, extension at 72℃ for 10 min, followed by incubation at 4℃. PCR products were detected by 1% agarose gel electrophoresis. Results are as follows: Figure 2 As shown, bands 1, 2, 3, 4, and 5 represent wild-type WT, plasmids containing mutants R247S, R247N, R247E, and R247A, respectively. The size of the target band amplified by the mutants is consistent with the theoretical size (6300 bp), indicating that the target band has been successfully amplified. The corresponding PCR products are purified and used for transformation experiments.

[0054] Add Dpn I restriction endonuclease to the PCR product, incubate at 37°C for 2 h to degrade the template, and then transform into E. coli JM109 competent cells. Spread the transformation product onto a plate containing 50 mg / mL Dpn I restriction endonuclease. -1The kanamycin was cultured on LB solid medium at 37°C for 10–12 h. Positive clones were then picked, inoculated onto LB medium, cultured overnight, and plasmids were extracted to prepare a wild-type plasmid (named pRSFDuet-0875-1647) and a 5-carboxy-2-pentenoyl-CoA reductase gene mutant plasmid (named pRSFDuet-0875-1647mut).

[0055] Add 5 μL each of pRSFDuet-0875-1647, pRSFDuet-0875-1647mut, and two other plasmids in the adipic acid synthesis pathway (pETDuet-0067-2399 containing the β-ketothiolase gene and the 3-hydroxyacyl-CoA dehydrogenase gene; and pCDuet-2567-2566 containing the 3-hydroxyacyl-CoA dehydrogenase gene and the adipicyl-CoA synthase gene) to 100 μL of BL21(DE3) competent cells, mix well, place on ice, incubate on ice for at least 30 min, then heat shock at 42°C for 90 s. After heat shock, quickly transfer to ice, incubate on ice for 5 min, then add 800 μL of LB medium and incubate on a shaker at 37°C and 200 r / min. One hour later, the cells were centrifuged at 3500 g for 3 minutes, 800 μL of supernatant was discarded, and the remaining liquid was used to suspend the bacterial cells. The cells were then evenly spread on solid LB plates containing Kan and incubated overnight at 37°C to obtain recombinant bacteria containing 5-carboxy-2-pentenoyl-CoA reductase mutant.

[0056] The construction process of plasmids ① pETDuet-0067-2399 and ② pCDuet-2567-2566 is as follows: Taking plasmid ① pETDuet-0067-2399 as an example, using the empty pETDuet-1 plasmid as a template, plasmid pETDuet-1 was linearized using primer pair pETDuet-F / pETDuet-R. The 0067-2399 fragment was amplified from the synthesized gene pUC57-0067-2399 using specific primers 0067-F and 2399-R. Then, homologous recombination of the two fragments was performed for transformation, identification, and sequencing to obtain pETDuet-0067-2399, making it more suitable for subsequent gene cloning and expression. Similarly, pCDuet-2567-2566 can be obtained by referring to the above method.

[0057] Example 3

[0058] This embodiment tests the adipic acid synthesis ability of the recombinant bacteria constructed in Example 2.

[0059] The recombinant bacteria were transferred to LB medium, and Kan, Amp, and Str antibiotics were added to final concentrations of 50 μg / mL, 100 μg / mL, and 50 μg / mL, respectively. The culture was incubated at 37°C and 220 rpm for 8-10 h to obtain a seed culture. The seed culture was then inoculated at a 2% inoculation rate into M9 medium (containing the same final concentrations of Kan, Amp, and Str). The culture was incubated at 37°C and 220 rpm until the OD reached 0.6-0.8. 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the culture was transferred to 30°C and fermented at 220 rpm. Samples were taken for analysis after the addition of IPTG (i.e., 0 h). Samples were taken every 6 h. The samples were centrifuged at 12000 g for 10 min at 4°C, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The samples were then analyzed using high-performance liquid chromatography (HPLC). (Example results are shown in...) Figure 3A As shown, this sample was fermented for 36 h by a strain containing mutant R247E. The concentration of adipic acid in the fermentation broth was analyzed. Detection conditions: The chromatographic column used was a Bio-Rad HPX-87H organic acid column; the mobile phase was 5 mM sulfuric acid solution; the detector was a differential detector; the flow rate of the mobile phase was 0.6 mL / min; and the detection temperature was 40℃.

[0060] The specific method for calculating adipic acid concentration is as follows: Different concentrations of adipic acid standards are detected, and a standard curve is plotted with the concentration of the adipic acid standard as the abscissa (X-axis) and the corresponding peak area as the ordinate (Y-axis). The standard curve equation is fitted using methods such as linear regression, generally in the form Y = kX + b, where k is the slope and b is the intercept. For example, the detection chromatogram of adipic acid standard with a concentration of 0.5 g / L is shown below. Figure 3B As shown. Substituting the peak area of ​​the fermentation sample into the standard curve equation (Y = 113771.2756X + 520.8597(R)), we get: 2 =0.9999), solve for the corresponding concentration value, which is the concentration of adipic acid in the fermentation sample.

[0061] Fermentation results of wild-type (WT) and different recombinant strains are as follows: Figure 4 As shown, the adipic acid yield of each strain reached its highest level 30 h after the addition of IPTG. In actual production, fermentation can be stopped near this time point and the product can be recovered. The adipic acid yield of each recombinant strain was higher than that of the wild strain, indicating that the 5-carboxy-2-pentenoyl-CoA reductase mutant designed in this invention can effectively increase the adipic acid yield, up to 2.2 g / L, which is 4.33 times higher.

[0062] In summary, this invention addresses the problem of low cofactor binding efficiency of 5-carboxy-2-pentenoyl-CoA reductase in the reverse adipic acid degradation pathway. Using the gene expressing 5-carboxy-2-pentenoyl-CoA reductase as the research object, rational prediction and site-directed mutagenesis experiments were conducted to optimize the enzyme's binding site structure, making it more adaptable to the characteristics of cofactors. This enhances the enzyme's cofactor binding ability, improves its catalytic activity in the reaction of 5-carboxy-2-pentenoyl-CoA to generate adipicyl-CoA, and ultimately increases the yield of the target product, adipic acid.

[0063] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A 5-carboxy-2-pentenoyl-CoA reductase mutant, characterized in that, The amino acid sequence of the mutant is mutated based on the sequence shown in SEQ ID NO.2, and the mutated site includes R247.

2. The 5-carboxy-2-pentenoyl-CoA reductase mutant according to claim 1, characterized in that, The mutation includes at least one of R247S, R247N, R247E, or R247A.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the 5-carboxy-2-pentenoyl-CoA reductase mutant as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3.

5. The recombinant vector according to claim 4, characterized in that, The starting vector for the recombinant vector includes a plasmid vector; Preferably, the plasmid vector includes any one of the following: pRSFDuet vector, pET vector, pETduet vector, pREST vector, pETDuet vector, or pCDuet vector.

6. A recombinant bacterium, characterized in that, The recombinant bacteria contain the nucleic acid molecule described in claim 3.

7. The recombinant bacteria according to claim 6, characterized in that, The starting strains of the recombinant bacteria include Escherichia coli and / or yeast; Preferably, the Escherichia coli includes Escherichia coli K-12 series strains and / or Escherichia coli B series strains; Preferably, the yeast includes any one of Saccharomyces cerevisiae, Isaac's orientalis, or Pichia pastoris; Preferably, the recombinant bacteria also express β-ketothiolytic enzyme gene, 3-hydroxyacyl-CoA dehydrogenase gene, 3-hydroxyadiacyl-CoA dehydrogenase gene, and adipicyl-CoA synthase gene.

8. The method for constructing the recombinant bacteria according to claim 6 or 7, characterized in that, The method for constructing the recombinant bacteria includes: The nucleic acid molecule of claim 3 is inserted into the genome of the starting strain; or the recombinant vector of claim 4 is introduced into the starting strain.

9. The use of the 5-carboxy-2-pentenoyl-CoA reductase mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4 or 5, or the recombinant bacteria of claim 6 or 7 in the synthesis of adipic acid.

10. A method for synthesizing adipic acid, characterized in that, The method includes: using the 5-carboxy-2-pentenoyl-CoA reductase mutant as described in claim 1 or 2 to participate in the catalytic synthesis reaction of adipic acid.

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