Formaldehyde polymerase mutant and use thereof

Abstract

The application provides a formaldehyde polymerase mutant and application thereof, and relates to the technical field of biological engineering and enzyme engineering. The application takes formaldehyde polymerase GALS-M4 as a parent, and a series of key amino acid residue pairs are obtained by protein engineering technology, and corresponding formaldehyde polymerase mutants are obtained. The experimental results show that, compared with the parent enzyme, the formaldehyde polymerase mutant with double-site mutation has significantly improved catalytic activity in the reaction of catalyzing formaldehyde to condense to generate hydroxyacetaldehyde. The application also discloses nucleic acid for coding the mutant, an expression vector containing the nucleic acid, a host cell, and application of the host cell in the field of C1 compound biological transformation and synthetic biology.

Description

Technical Field

[0001] This application relates to the fields of bioengineering and enzyme engineering technology, and in particular to a formaldehyde polymerase mutant and its applications. Background Technology

[0002] Formaldehyde is an important one-carbon (C1) compound, serving as both a common raw material in industrial production and an intermediate product in certain biological metabolic processes. Enzymatic polymerization of formaldehyde to generate high-value-added multi-carbon compounds (such as hydroxyacetaldehyde and dihydroxyacetone) is a crucial pathway for carbon fixation in synthetic biology, holding significant importance for sustainable biomanufacturing. Formaldehyde polymerase (GALS) catalyzes the condensation of two formaldehyde molecules to form hydroxyacetaldehyde (GALD). However, currently known natural formaldehyde polymerases often suffer from low catalytic efficiency, insufficient substrate affinity, or poor stability, making it difficult to meet the demands of large-scale industrial applications. Therefore, modifying formaldehyde polymerases through protein engineering to obtain mutants with high catalytic activity is a key technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0003] In view of the problems of the prior art, the object of the present invention is to provide a formaldehyde polymerase mutant with significantly enhanced catalytic activity.

[0004] To address the aforementioned issues, the inventors employed a rational design strategy based on protein three-dimensional structure analysis. By deeply analyzing the three-dimensional model structure of the parental enzyme formaldehyde polymerase GALS-M4 (SEQ ID No. 1), a dual-site mutation library was constructed targeting these specific paired sites using high-fidelity enzymatic DNA synthesis technology. Combined with GFP fluorescence internal control normalization and high-throughput mass spectrometry detection technology, a series of high-performance mutants were screened and obtained.

[0005] On one hand, this application provides a formaldehyde polymerase mutant, said formaldehyde polymerase mutant comprising any one of the following A1)-A3): A1) A protein containing one or more mutations relative to the following sites of a reference sequence: positions 9, 35, 43, 44, 45, 85, 86, 383, 386, 387, 389, 395, 462, and 463, wherein each amino acid position is numbered by a reference sequence defined by the reference sequence, which comprises the amino acid sequence shown in SEQ ID No. 1; preferably, it contains multiple mutations relative to the following sites of the reference sequence; more preferably, it contains two mutations relative to the following sites of the reference sequence; A2) includes proteins obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the formaldehyde polymerase mutant described in A1), which have 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% identity with and function the same as the protein described in A1. A3) is a fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0006] Preferably, as those skilled in the art will understand, the tag may include a "tag" that facilitates purification, such as a histidine (HIS) tag, a glutathione-S-transferase tag (GST), a maltose-binding protein tag (MBP), a calmodulin-binding peptide tag (CBP), etc. Proteins tagged in this way can be easily purified, for example, from conditioned media by chelation chromatography or affinity chromatography.

[0007] Further, the formaldehyde polymerase mutant A1) includes any one or more of the following B1)-B14): B1) The tyrosine residue at position 9 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the tyrosine residue at position 9 of the reference sequence is mutated to a lysine residue. B2) The phenylalanine residue at position 35 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the phenylalanine residue at position 35 of the reference sequence is mutated to a cysteine ​​residue. B3) The leucine residue at position 43 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the leucine residue at position 43 of the reference sequence is mutated to an asparagine residue. B4) The alanine residue at position 44 of the reference sequence is mutated to one of the following residues: glycine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the alanine residue at position 44 of the reference sequence is mutated to a tyrosine residue. B5) The leucine residue at position 45 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, and glutamic acid; preferably, the leucine residue at position 45 of the reference sequence is mutated to one of the following residues: aspartic acid or tryptophan. B6) The alanine residue at position 85 of the reference sequence is mutated to one of the following residues: glycine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the alanine residue at position 85 of the reference sequence is mutated to tryptophan. B7) The arginine residue at position 86 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, histidine, aspartic acid, or glutamic acid; preferably, the arginine residue at position 86 of the reference sequence is mutated to a proline residue. B8) The methionine residue at position 383 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the methionine residue at position 383 of the reference sequence is mutated to one of the following residues: glutamine, glutamic acid, isoleucine, cysteine, or threonine. B9) The arginine residue at position 386 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, histidine, aspartic acid, or glutamic acid; preferably, the arginine residue at position 386 of the reference sequence is mutated to a leucine residue. B10) The leucine residue at position 387 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the leucine residue at position 387 of the reference sequence is mutated to a phenylalanine residue. B11) The methionine residue at position 389 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, arginine, histidine, aspartic acid, and glutamic acid; preferably, the methionine residue at position 389 of the reference sequence is mutated to one of the following residues: isoleucine or arginine. B12) The tyrosine residue at position 395 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid; preferably, the tyrosine residue at position 395 of the reference sequence is mutated to an aspartic acid residue. B13) The arginine residue at position 462 of the reference sequence is mutated to one of the following residues: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, glutamine, tyrosine, lysine, histidine, aspartic acid, or glutamic acid; preferably, the arginine residue at position 462 of the reference sequence is mutated to a cysteine ​​residue. B14) The tryptophan residue at position 463 of the reference sequence is mutated to one of the following residues: glycine residue, alanine residue, valine residue, leucine residue, isoleucine residue, methionine residue, proline residue, phenylalanine residue, serine residue, threonine residue, cysteine ​​residue, asparagine residue, glutamine residue, tyrosine residue, lysine residue, arginine residue, histidine residue, aspartic acid residue, and glutamic acid residue; preferably, the tryptophan residue at position 463 of the reference sequence is mutated to one of the following residues: glutamine residue and tyrosine residue.

[0008] Further, the A1) formaldehyde polymerase mutant includes any one or more of the following C1)-C11): C1) Proteins (M383Q, M389I) in which the methionine residue at position 383 is mutated to a glutamine residue and the methionine residue at position 389 is mutated to an isoleucine residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:3, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:14. C2) Proteins (M383E, L387F) in which the methionine residue at position 383 is mutated to a glutamic acid residue and the leucine residue at position 387 is mutated to a phenylalanine residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:4, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:15. C3) Proteins (M383I, R386L) in which the methionine residue at position 383 is mutated to isoleucine and the arginine residue at position 386 is mutated to leucine relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:5, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:16. C4) Proteins (L43N, L45W) in which the leucine residue at position 43 is mutated to an asparagine residue and the leucine residue at position 45 is mutated to a tryptophan residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:6, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:17. C5) A protein (R462C, W463Y) in which the arginine residue at position 462 is mutated to a cysteine ​​residue and the tryptophan residue at position 463 is mutated to a tyrosine residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:7, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:18. C6) A protein (M383C, Y395D) in which the methionine residue at position 383 is mutated to a cysteine ​​residue and the tyrosine residue at position 395 is mutated to an aspartic acid residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:8, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:19. C7) A protein (Y9K, F35C) in which the tyrosine residue at position 9 of the reference sequence is mutated to a lysine residue and the phenylalanine residue at position 35 is mutated to a cysteine ​​residue; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:9, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:20. C8) A protein (A85W, R86P) in which the alanine residue at position 85 of the reference sequence is mutated to a tryptophan residue and the arginine residue at position 86 is mutated to a proline residue; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:10, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:21. C9) A protein (A44Y, L45D) in which the alanine residue at position 44 is mutated to a tyrosine residue and the leucine residue at position 45 is mutated to an aspartic acid residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:11, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:22. C10) is a protein (R462C, W463Q) in which the arginine residue at position 462 is mutated to a cysteine ​​residue and the tryptophan residue at position 463 is mutated to a glutamine residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:12, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:23; C11) Proteins (M383T, M389R) in which the methionine residue at position 383 is mutated to a threonine residue and the methionine residue at position 389 is mutated to an arginine residue relative to the reference sequence; preferably, the amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO:13, and the nucleotide sequence encoding the formaldehyde polymerase mutant is shown in SEQ ID NO:24.

[0009] Further, the formaldehyde polymerase mutant comprises the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and / or SEQ ID NO.13.

[0010] It is understandable that those skilled in the art can select appropriate gene editing systems and gene editing methods to obtain the above-mentioned mutants based on the actual situation.

[0011] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following D1)-D6): D1) A nucleic acid molecule encoding the formaldehyde polymerase mutant; D2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1) and / or the expression cassette described in D2); D4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in D1), the expression cassette described in D2), and / or the recombinant vector described in D3; D5) Recombinant cells containing the nucleic acid molecule described in D1), the expression cassette described in D2), and / or the recombinant vector described in D3); D6) Whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in D1), the expression cassette described in D2), the recombinant vector described in D3), the recombinant microorganism described in D4), and / or the recombinant cell described in D5).

[0012] The expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make conventional selections according to the actual situation, as long as the expression of D1 nucleic acid molecules can be completed. No further restrictions are placed on the structure and composition of the expression cassette here.

[0013] The recombinant vectors described herein refer to vectors capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA manipulation and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed-circular plasmids. Vectors can be self-replicating vectors (i.e., complete structures existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. Vectors can contain any mechanism that ensures self-replication. Alternatively, a vector is one that, when introduced into a host cell, integrates into the genome and replicates along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used, as those skilled in the art can choose according to the specific circumstances; no excessive limitations are imposed here.

[0014] In one alternative implementation, the carrier may be pET-28a.

[0015] Furthermore, the nucleic acid molecule described in D1) includes any one of the following E1)-E5): E1) encodes the nucleic acid molecule of the formaldehyde polymerase mutant; E2) Nucleic acid molecules encoding the amino acid sequences of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13; E3) Nucleic acid molecules with nucleotide sequences such as SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 or SEQ ID No. 24; E4) and any of the nucleic acid molecules described in E1)-E3) have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9% identity, and encode the nucleic acid molecule of the formaldehyde polymerase mutant; E5) hybridizes with any of the nucleic acid molecules described in E1)-E3) under stringent conditions and encodes the genomic DNA molecule of the formaldehyde polymerase mutant.

[0016] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain to construct recombinant microorganisms, as long as they can complete the expression of the mutants described in this application. No specific strains are limited here.

[0017] Furthermore, the recombinant microorganism is one or more of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

[0018] Preferably, the recombinant microorganism is Escherichia coli.

[0019] In one alternative embodiment, the recombinant microorganism is Escherichia coli BL21(DE3).

[0020] The whole-cell catalyst may include suspensions, metabolites, extracts, etc. of recombinant microorganisms or recombinant cells.

[0021] In one optional implementation, the recombinant vector and recombinant cells contain an resistance selection marker gene. Those skilled in the art can choose according to the actual situation. This application does not impose mandatory limitations on the resistance selection marker gene.

[0022] A resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or a protrophic auxotrophic phenotype. Examples of bacterial selection markers include the dal gene in Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline.

[0023] It is understood that those skilled in the art can select appropriate gene editing systems and gene editing methods to obtain the above-mentioned biological materials based on the actual situation.

[0024] On the other hand, this application also provides a method for preparing the formaldehyde polymerase mutant, the method comprising: producing the formaldehyde polymerase mutant by fermentation using the recombinant microorganism.

[0025] The fermentation can be carried out using conventional methods.

[0026] Preferably, the preparation method includes the following steps: Step 1: Construct recombinant microorganisms expressing the formaldehyde polymerase mutant; Step 2: Fermentation culture of the recombinant microorganisms.

[0027] More preferably, the fermentation conditions include: culturing at 25℃-40℃ and 600-800 rpm until OD. 600 To induce expression, reduce the temperature to 10℃-20℃, add IPTG to a final concentration of 0.1-0.5 mM, and induce expression for 12-24 h; more preferably, culture at 37℃ and 700 rpm until OD. 600 The concentration was set to 0.6-0.8, the temperature was lowered to 16℃, IPTG was added to a final concentration of 0.5 mM, and expression was induced for 16-18 h.

[0028] More preferably, the fermentation medium is 2YT liquid medium.

[0029] More preferably, the preparation method further includes an activation step.

[0030] More preferably, the preparation method includes the following steps: Step 1: Construct recombinant microorganisms expressing the formaldehyde polymerase mutant; Step 2: Incubate the recombinant microorganisms in an activation medium at 25℃-40℃ and 100-300 rpm for 8-48 h to obtain seed culture; Step 3: Inoculate the seed culture into the fermentation medium at an inoculum volume of 1%-5% (v / v). The fermentation conditions include: culturing at 25℃-40℃ and 600-800 rpm until OD (dose-to-temperature ratio) is reached. 600To obtain a concentration of 0.4-1, lower the temperature to 10℃-20℃, add IPTG to a final concentration of 0.1-0.5 mM, and induce expression for 12-24 h.

[0031] More preferably, Step 1: Construct recombinant microorganisms expressing the formaldehyde polymerase mutant; Step 2: Incubate the recombinant microorganisms in an activation medium at 37°C and 200 rpm for 8-48 h to obtain seed culture; Step 3: Inoculate the seed culture into the fermentation medium at a 2% inoculation rate (v / v, volume ratio). The fermentation conditions include: culturing at 37°C and 700 rpm until OD. 600 The concentration was set to 0.6-0.8, the temperature was lowered to 16℃, IPTG was added to a final concentration of 0.5 mM, and expression was induced for 16-18 h.

[0032] More preferably, the activation medium is LB medium.

[0033] Those skilled in the art can use known purification methods to purify the above fermentation products to obtain formaldehyde polymerase mutants, and no specific limitations are made here.

[0034] On the other hand, this application also provides the application of the formaldehyde polymerase mutant or the biomaterial in catalyzing formaldehyde polymerization; preferably, the formaldehyde polymerization reaction includes catalyzing the formation of hydroxyacetaldehyde from formaldehyde.

[0035] More preferably, the reaction conditions for the formaldehyde polymerization reaction include: reacting at 30℃-40℃ for 1-5 h; more preferably, reacting at 37℃ for 2 h.

[0036] The formaldehyde polymerization reaction includes the polymerization of formaldehyde to generate multi-carbon compounds; preferably, the multi-carbon compounds include hydroxyacetaldehyde, dihydroxyacetone, etc.

[0037] More preferably, the formaldehyde polymerization reaction comprises the condensation of two formaldehyde molecules to generate hydroxyacetaldehyde.

[0038] Those skilled in the art can select the amount or concentration of the formaldehyde polymerase mutant or the biomaterial to be added based on the actual situation, and no specific limitation is made here.

[0039] More preferably, the reaction further includes the cofactor TPP, magnesium ions, and a buffer solution.

[0040] In this catalytic reaction, magnesium ions (Mg²⁺) + Magnesium is used to maintain the stability of the enzyme protein structure. In the experiment, any soluble magnesium salt (such as MgSO4, MgCl2, etc.) can be selected as the magnesium source, as long as it does not affect the overall conversion efficiency.

[0041] More preferably, the buffer solution may be a potassium phosphate buffer solution.

[0042] The main function of a buffer solution is to provide a suitable pH environment and ion concentration conditions for the system. In practice, phosphate buffers such as potassium phosphate and sodium phosphate, or commonly used buffers such as TEA, Tris, and HEPES can all be used, and no specific restrictions are made here.

[0043] On the other hand, this application also provides the use of the formaldehyde polymerase mutant or the biomaterial in the preparation of hydroxyacetaldehyde and / or products containing hydroxyacetaldehyde and / or products containing hydroxyacetaldehyde structural units.

[0044] On the other hand, this application also provides a product comprising the formaldehyde polymerase mutant or the biomaterial.

[0045] The product of this application may also contain excipients, which may be appropriate solvents, solubilizers, co-solvents, emulsifiers, osmotic pressure regulators, stabilizers, pH regulators, buffers, diluents, protectants, etc.

[0046] The product of this application can be prepared by a general method, wherein one or more protective agents or carriers can be added to form a dosage form commonly used in the art.

[0047] The product may be a catalyst for the preparation of hydroxyacetaldehyde and / or a catalyst for the polymerization of formaldehyde.

[0048] On the other hand, this application also provides the use of the product in the preparation of hydroxyacetaldehyde and / or in the catalytic polymerization of formaldehyde.

[0049] On the other hand, this application also provides a method for improving formaldehyde polymerase activity, the method comprising: mutating one or more sites from positions 9, 35, 43, 44, 45, 85, 86, 383, 386, 387, 389, 395, 462, and 463 of a reference sequence, wherein the reference sequence comprises the amino acid sequence shown in SEQ ID No. 1; preferably, multiple sites in the reference sequence are mutated; more preferably, two sites in the reference sequence are mutated; even more preferably, the mutated sites are key amino acid residue pairs with spatial interactions near the active site and / or substrate channel.

[0050] The present invention has the following beneficial effects: This invention uses formaldehyde polymerase GALS-M4 (SEQ ID No.1) as the parent and obtains a series of key amino acid residue pairs and corresponding formaldehyde polymerase mutants through protein engineering technology.

[0051] Experimental results show that, compared with the parent enzyme, the formaldehyde polymerase mutant with two site mutations of the present invention exhibits significantly enhanced catalytic activity in the reaction of formaldehyde condensation to hydroxyacetaldehyde, with the optimal mutant showing a relative enzyme activity 6.47 times higher than that of the parent. The present invention also discloses the nucleic acid encoding the above mutant, the expression vector containing the nucleic acid, the host cell, and its applications in the biotransformation of C1 compounds and synthetic biology.

[0052] This invention demonstrates the effectiveness of the dual-site synergistic mechanism. The specific pairwise mutations (such as charge complementarity or volume complementarity) of this invention successfully remodeled the enzyme microenvironment, resulting in mutants with high catalytic activity. Such high effects are difficult to achieve with random mutations alone. This proves the superiority of the strategy of improving enzyme activity by manipulating spatial interaction networks, and provides a new modification approach for the field of enzyme engineering.

[0053] The highly active mutant of this invention makes it possible to achieve efficient formaldehyde polymerization under low enzyme load, which will significantly reduce the metabolic burden and production cost of C1 compound biotransformation, and provide a powerful biocatalyst for using formaldehyde as a raw material to synthesize high value-added chemicals, with broad application prospects. Attached Figure Description

[0054] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the 3D structure of the formaldehyde polymerase GALS-M4 protein, where red indicates the location of the mutation sites screened in this invention in the protein structure; Figure 2 This is a plasmid map, where GALS Mutant is the DNA fragment encoding the formaldehyde polymerase mutant. Figure 3 A bar chart showing the relative activities of the 10 formaldehyde polymerase mutants screened in this invention compared with the parental formaldehyde polymerase GALS-M4. Detailed Implementation

[0055] Technical terms: Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0056] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0057] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0058] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0059] Recombinant microorganisms: bacterial cell lines that use genetic engineering methods to achieve efficient expression of foreign genes.

[0060] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0061] The term “and / or” as used in this invention refers to and covers any and all possible combinations of one or more of the related listed items, as well as the absence of a combination when interpreted in an alternative manner (“or”).

[0062] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0063] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. 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 application pertains. Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0064] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0065] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0066] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0067] In this specification, mutations of amino acids are referred to as "original amino acid, site, substituted amino acid". For example, the mutation of methionine M at position 383, counting sequentially from the N (nitrogen) end to the C (carbon) end, to glutamine Q is represented as M383Q.

[0068] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0069] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0070] LB medium: Tryptone 10 g / L, Yeast Extract 5 g / L, NaCl 10 g / L, balance water.

[0071] 2YT liquid culture medium (containing kanamycin): Tryptone 16 g / L, Yeast Extract 10 g / L, NaCl 5 g / L, Kanamycin 0.05 g / L, with the remainder being water.

[0072] Example 1: Construction of mutant libraries This embodiment uses formaldehyde polymerase GALS-M4 as the parent and obtains a series of formaldehyde polymerase mutants through protein engineering technology. The steps are as follows: (1) Parental sequence: The formaldehyde polymerase GALS-M4 was used as the parent, and its amino acid sequence is shown in SEQ ID No.1.

[0073] (2) Library design: Based on the analysis of the protein structure of formaldehyde polymerase GALS-M4, key amino acid sites located near its active site and substrate channel (such as positions 9, 35, 43, 44, 45, 85, 86, 383, 386, 387, 389, 395, 462, and 463, etc.) were selected. Figure 1 As shown, pairwise mutation designs with different combinations of mutation directions were carried out.

[0074] (3) Library preparation: A DNA fragment (approximately 130-200 bp in length) encoding the formaldehyde polymerase mutant containing the above-mentioned mutation sites was synthesized using enzymatic DNA synthesis technology, and then inserted into the expression vector pET-28a containing the GFP reporter gene using a seamless cloning method. NdeI and XhoI Between the restriction enzyme sites, a formaldehyde polymerase mutant expression plasmid library was constructed, such as... Figure 2 As shown.

[0075] The construction method included: Zhonghe Gene Co., Ltd. was commissioned to synthesize the linear mutant gene fragment (insert) encoding the formaldehyde polymerase mutant using enzymatic DNA synthesis technology. Homologous recombination (seamless cloning) of the mutant gene fragment and the linearized pET-28a-GFP plasmid backbone was performed using the OKClon DNA ligation kit (Hunan Aike Rui Biotechnology Co., Ltd., catalog number AG11807 / AG11808). 50 ng of the linearized pET-28a-GFP plasmid was added to the reaction system as the backbone, followed by 50 ng of the enzymatically synthesized linear mutant gene fragment. ddH2O was added to bring the volume to 6 μL, followed by 4 μL of 2.5× OK Clon premixed buffer. The mixture was then pipetted and placed in a PCR instrument at 37℃ for 20 min to obtain the ligation product. The product was removed from the -80℃ freezer 5 min beforehand. E. coli BL21(DE3) competent cells. All of the above ligation product was aspirated and slowly injected into 100 μL of the solution with stirring. E. coliIn a BL21(DE3) competent cell system, the cells were incubated on ice for 25 min, heat-shocked at 42°C for 45 s, and immediately cooled on ice for 2 min. 700 μL of antibiotic-free LB medium was added, and the cells were revived at 37°C and 200 rpm for 1 h. The revived bacterial culture was transferred to 96-well plates (final volume 1 mL, containing a final kanamycin concentration of 100 μg / mL) and cultured overnight at 37°C with a shaking incubator at 200 rpm to obtain a formaldehyde polymerase mutant expression plasmid library.

[0076] The nucleotide sequence of the GALS-M4 enzyme encoding gene is shown in SEQ ID No. 2. The vector for expressing the GALS-M4 enzyme is formed by inserting the GALS-M4 enzyme encoding gene (SEQ ID No. 2) into a pET-28a vector containing a GFP reporter gene. NdeI and XhoI The vector obtained between the restriction enzyme sites was named pET-28a-GALS-M4. pET-28a-GALS-M4 and engineered bacteria containing pET-28a-GALS-M4 were constructed using the same method as described above as controls.

[0077] Example 2: Protein Expression and Screening Process (1) Induced expression of mutant proteins 20 μL of the initial bacterial culture (formaldehyde polymerase mutant expression plasmid library or engineered bacteria containing pET-28a-GALS-M4) from overnight culture in Example 1 was transferred to a 96-well plate containing 1 mL of fresh 2YT liquid medium (containing kanamycin). The plate was incubated at 37°C and 700 rpm until OD200 was reached. 600 The concentration was approximately 0.6-0.8 (normal culture time is about 3-4 h). The temperature was then lowered to 16℃, and IPTG was added to a final concentration of 0.5 mM to induce expression for 16-18 h.

[0078] (2) Sample pretreatment and enzymatic reaction Centrifuge the 96-well plate after induction of expression in step (1) at 3500 rpm for 5 min. After centrifugation, invert the plate to remove the supernatant. Add 150 μL of potassium phosphate buffer (50 mM, pH 7.4) to each well using a multi-pipette, resuspend the cells by pipetting, centrifuge again (3500 rpm, 5 min), and invert the plate to remove the supernatant. Repeat the washing step once to thoroughly remove any culture medium residue and avoid interference with subsequent detection. Add 200 μL of reaction solution to each well using a multi-pipette. The reaction solution consists of: substrate formaldehyde (final concentration 6 g / L), cofactor TPP (0.5 mM), and MgSO4 (5 mM), dissolved in 150 μL of 50 mM potassium phosphate buffer. Place the 96-well plate in a 37°C incubator for 2 h.

[0079] (3) Dual-modal high-throughput detection Fluorescence assay (internal control): Before the enzymatic reaction in step (2), the 96-well plate induced by expression in step (1) was centrifuged at 3500 rpm for 5 min. After centrifugation, the plate was inverted to remove the supernatant. 150 μL of potassium phosphate buffer (50 mM, pH 7.4) was added to each well using a pipette, and the bacterial cells were resuspended evenly by pipetting. 5 μL of the resuspended bacterial solution was added to 95 μL of double-distilled water, and after washing evenly, 100 μL of the mixture was placed in a black microplate. The GFP fluorescence intensity value (Excitation 485 nm / Emission 520 nm) of each well was read using a microplate reader and recorded as Fluorescence Value, which was used to characterize the expression level of the mutant protein in each well. Formaldehyde polymerase GALS-M4 was used as the parental control group.

[0080] Mass spectrometry detection (product): After the enzymatic reaction in step (2) is completed, the bacterial cells are precipitated by centrifugation in the 96-well plate. 40-50 μL of the supernatant is directly transferred to a shallow-well 384-well plate compatible with AEMS. High-throughput injection detection is performed using a SCIXE AE-MS (Acoustic Ejection Mass Spectrometry, Echo MS) system. The characteristic peak area of ​​the reaction product hydroxyacetaldehyde (GALD) is quantitatively detected and recorded as the Metric Value (Area).

[0081] Data Analysis: The normalized activity of each mutant was calculated using the formula: Specific Activity = Metric Value (Area) / Fluorescence Value. The mutants were then compared with the parental control (formaldehyde polymerase GALS-M4) which exhibited the highest relative activity on the same 96-well plate. The relative parental fold was calculated using the formula: Relative Parental Fold = Sample Relative Activity / Max (Parental Relative Activity). Mutants showing significantly enhanced activity compared to GALS-M4 were screened. After screening and re-screening, 10 formaldehyde polymerase mutants with significantly superior activity to the parent were obtained. Specific mutation sites and activity data are shown in Table 1, and graphs were plotted from Table 1. Figure 3 .

[0082] Table 1: Mutation sites and activity data of highly active GALS mutants and control mutants

[0083] As shown in Table 1, this embodiment screened and obtained 10 enzyme mutants with relatively increased activity compared to the parental formaldehyde polymerase GALS-M4: double mutants of M383Q and M389I, M383E and L387F, M383I and R386L, L43N and L45W, R462C and W463Y, M383C and Y395D, Y9K and F35C, A85W and R86P, A44Y and L45D, and R462C and W463Q. Multiple mutation sites and their combinations that can enhance the activity of formaldehyde polymerase GALS-M4 were also identified.

[0084] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A formaldehyde polymerase mutant, characterized in that, The formaldehyde polymerase mutant is a protein in which the methionine residue at position 383 is mutated to a glutamine residue and the methionine residue at position 389 is mutated to an isoleucine residue relative to the reference sequence, wherein the amino acid positions are numbered by the reference sequence as shown in SEQ ID No.

1.

2. The formaldehyde polymerase mutant according to claim 1, characterized in that, The amino acid sequence of the formaldehyde polymerase mutant is shown in SEQ ID NO.

3.

3. A biomaterial, characterized in that, The biomaterial includes any one of the following D1)-D6): D1) A nucleic acid molecule, said nucleic acid molecule encoding the formaldehyde polymerase mutant of claim 1 or 2; D2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1) and / or the expression cassette described in D2); D4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in D1), the expression cassette described in D2), and / or the recombinant vector described in D3; D5) Recombinant cells containing the nucleic acid molecule described in D1), the expression cassette described in D2), and / or the recombinant vector described in D3); D6) Whole-cell catalyst, wherein the whole-cell catalyst contains the nucleic acid molecule described in D1), the expression cassette described in D2), the recombinant vector described in D3), the recombinant microorganism described in D4), and / or the recombinant cell described in D5).

4. The biomaterial as described in claim 3, characterized in that, The nucleic acid molecule described in D1 is shown in SEQ ID No.

14.

5. The biomaterial according to claim 3, characterized in that, The recombinant microorganism is one or more of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

6. The application of the formaldehyde polymerase mutant as described in claim 1 or 2, or the biomaterial as described in any one of claims 3-5, in the catalytic formaldehyde polymerization reaction.

7. The application according to claim 6, characterized in that, The formaldehyde polymerization reaction includes catalyzing the formation of hydroxyacetaldehyde from formaldehyde.

8. The use of the formaldehyde polymerase mutant as described in claim 1 or 2, or the biomaterial as described in any one of claims 3-5, in the preparation of hydroxyacetaldehyde and / or products containing hydroxyacetaldehyde and / or products containing hydroxyacetaldehyde structural units.

9. A product characterized in that, The product includes the formaldehyde polymerase mutant as described in claim 1 or 2, or the biomaterial as described in any one of claims 3-5.

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