Formaldehyde dehydrogenase mutants and uses thereof

Abstract

The application discloses a formaldehyde lyase mutant and application thereof. The formaldehyde lyase mutant provided by the application can regulate the generation proportion of a two-carbon and three-carbon product generated by catalyzing formaldehyde condensation, has improved activity of catalyzing formaldehyde condensation into glycolaldehyde, and reduced activity of catalyzing formaldehyde condensation into 1,3-dihydroxyacetone, compared with formaldehyde lyase.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a formaldehyde enzyme mutant and its uses. Background Technology

[0002] Formaldehyde is a hot topic in the research of one-carbon compounds. It is widely available and inexpensive, and can be obtained from compounds such as CO2, methanol, and methane through chemical or biosynthetic methods, making it an ideal substrate for artificial carbon fixation pathways. Therefore, utilizing enzyme elements that can catalyze the conversion of one-carbon formaldehyde into new multi-carbon products to achieve formaldehyde conversion and utilization has significant industrial application value.

[0003] In 2015, researchers including David Baker, through rational design, first modified benzaldehyde condensate (PDB ID: 2AG0) to obtain a novel formolase (FLS) (PDB ID: 4QQ8) that can catalyze the formation of a new multi-carbon product from the one-carbon compound formaldehyde. This enzyme, based on the benzaldehyde lyase backbone and modified through active site modification, can catalyze the condensation of three formaldehyde molecules to produce one molecule of 1,3-dihydroxyacetone (DHA) (Siegel JB, et al. Proc NatlAcad Sci USA, 2015, 112: 3704-3709). Cai et al. modified FLS using directed evolution, obtaining the three-point mutant FLS-M3 (I28L / T90L / N283H), which increased the activity of FLS in catalyzing the production of 1,3-dihydroxyacetone (Cai T, et al. Science, 2021, 373: 1523-1527). However, despite various modifications to FLS using protein engineering techniques to enhance its ability to generate the three-carbon product 1,3-dihydroxyacetone, its efficiency in catalyzing the two-carbon product glycolaldehyde (GA) remains low, becoming a key bottleneck restricting the further application of this enzyme in high-efficiency carbon conversion systems.

[0004] Ethanolaldehyde, as the simplest α-hydroxyaldehyde, is an important C2 platform compound for the synthesis of high-value-added fine chemicals such as antiviral drugs, amine compounds, and fabric softeners. Previous studies have found that erythritol (ERY), a long-chain four-carbon product condensed from formaldehyde, can be obtained by converting the two-carbon product erythritol produced from formaldehyde using FLS catalysis. Volker Sieber et al. obtained mutants from the H29 and Q113 sites of FLS. Two double-site mutants, FLS_A3 (H29D / Q113C) and FLS_B2 (H29I / Q113S), successfully co-catalyzed formaldehyde to obtain the four-carbon product erythritol and the three-carbon product 1,3-dihydroxyacetone, with a ratio of 0.76, significantly increasing the proportion of erythritol in the product (Güner S, et al. Green Chem, 2021, 23: 6583-6590). Although this variant combination improves its activity in generating four-carbon products, the formation rate of glycolaldehyde, a key intermediate, directly affects the synthesis efficiency of subsequent four-carbon products. Its limited formation will further restrict the flux and yield of the entire carbon conversion pathway. Currently, the multi-carbon products obtained by formaldehyde lyase catalysis are mainly the three-carbon product 1,3-dihydroxyacetone. Therefore, improving the activity of FLS in catalyzing the synthesis of two-carbon products, especially by making the FLS-catalyzed products more selective towards two-carbon products, and thus solving the problem of controlling the ratio of two-carbon to three-carbon and other multi-carbon products, still requires further in-depth research.

[0005] Therefore, developing FLS variants that can efficiently catalyze the formation of ethanolaldehyde from formaldehyde and have adjustable product selectivity is of great significance for improving the conversion rate of C2 platform compounds and optimizing the carbon flow distribution of the one-carbon formaldehyde assimilation pathway. It can also lay a solid foundation for constructing an efficient industrial synthesis route for multi-carbon compounds starting from one-carbon formaldehyde. Summary of the Invention

[0006] In order to solve one of the above-mentioned technical problems in the prior art, the present invention provides a formaldehyde enzyme mutant and its uses.

[0007] The first aspect of the present invention provides a formaldehyde enzyme (FLS) mutant comprising an amino acid sequence having at least 95% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with the amino acid sequence of FLS as shown in SEQ ID NO:2. The formaldehyde enzyme mutant contains mutations at at least three sites: I28, T90, and N283. The formaldehyde enzyme mutant contains at least aspartic acid or leucine at position 28, leucine at position 90, and histidine at position 283.

[0008] The formaldehyde enzyme mutant provided in this invention has, relative to the FLS, increased activity in catalyzing the condensation of formaldehyde into glycolaldehyde and decreased activity in catalyzing the condensation of formaldehyde into 1,3-dihydroxyacetone.

[0009] In some embodiments, the formaldehyde enzyme mutant also includes mutations at the P43 and / or L112 sites.

[0010] In some embodiments, the formaldehyde enzyme mutant further includes serine at position 43 and / or threonine at position 112.

[0011] In some embodiments, the formaldehyde enzyme mutant comprises amino acid substitutions for I28D, T90L, and N283H; or, The formaldehyde enzyme mutant contains amino acid substitutions of I28L, T90L, L112T, and N283H; or, The formaldehyde enzyme mutant contains amino acid substitutions of I28L, P43S, T90L, L112T, and N283H.

[0012] In some embodiments, the formaldehyde enzyme mutant comprises an amino acid sequence having at least 95% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) with the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0013] In some embodiments, the amino acid sequence of the formaldehyde enzyme mutant is shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0014] It is understood that the formaldehyde enzyme mutant may further include an optional tag sequence for co-expression and / or purification. In some embodiments, the tag sequence is selected from at least one of His tag, FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA; more specifically, a His tag.

[0015] A second aspect of the present invention provides biomaterials related to formaldehyde enzyme mutants, said biomaterials comprising any one of (a1)-(a5): (a1) A nucleic acid molecule encoding the formaldehyde enzyme mutant according to claim 1 or 2 (a2) A vector containing the nucleic acid molecule described in (a1); (a3) A host cell that contains the nucleic acid molecule described in (a1) or the vector described in (a2); (a4) A cell culture comprising the host cells cultured under conditions suitable for the expression of the formaldehyde enzyme mutant in the host cells described in (a3); (a5) A recombinant microorganism comprising the nucleic acid molecule described in (a1) or the vector described in (a2).

[0016] In some embodiments, the vector is an expression vector, including prokaryotic or eukaryotic expression vectors. In some embodiments, the recombinant microorganism is an endogenous microorganism containing an operon encoding a formaldehyde detoxification system, wherein the operon is missing or inactivated. This system is also known as a glutathione-dependent formaldehyde oxidation system. Inactivation or deletion of the operon prevents formaldehyde from being consumed by the formaldehyde detoxification system, ensuring the substrate (formaldehyde) utilization rate of the formaldehyde enzyme mutant, thereby increasing the yield of the target product. Specifically, inactivation of the operon should prevent formaldehyde from being oxidized to formic acid. In this invention, the microorganism with a missing or inactivated operon in the formaldehyde detoxification system refers to a microorganism in which the core gene cluster frmRAB in the formaldehyde detoxification system is knocked out. frmRAB consists of three cis-arranged genes: frmR, frmA, and frmB.

[0017] In some embodiments, the eukaryotic expression vector includes yeast expression vectors, mammalian expression vectors, or insect expression vectors.

[0018] In some embodiments, the host cell is selected from prokaryotic cells and eukaryotic cells.

[0019] In some embodiments, the prokaryotic cells include *Escherichia coli*, *Bacillus subtilis*, *Pseudomonas*, and *Vibrio*. In some embodiments, the prokaryotic cells are *Escherichia coli*, specifically *Escherichia coli* in which the formaldehyde detoxification operon is missing or inactivated.

[0020] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells (such as CHO cells, HEK293 cells, HeLa cells), insect cells, etc.

[0021] In some embodiments, the microorganism is Escherichia coli, specifically Escherichia coli in which the formaldehyde detoxification operon is missing or inactivated.

[0022] A third aspect of the present invention provides a method for producing a formaldehyde enzyme mutant, comprising: The recombinant host cells are cultured in a culture medium under conditions suitable for the expression of the formaldehyde enzyme mutant in the host cells according to the second aspect of the present invention; and the formaldehyde enzyme mutant is obtained from the cell culture.

[0023] A fourth aspect of the present invention provides a composition comprising: (b1) the formaldehyde enzyme mutant of the first aspect of the present invention or the cell culture of the second aspect of the present invention; and (b2) Formaldehyde, thiamine pyrophosphate, Mg 2+ .

[0024] In some embodiments, the composition further comprises a buffer system with a pH of 7.0-8.0 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0).

[0025] In some embodiments, the buffer system is selected from phosphate buffer, HEPES buffer, or Tris-HCl buffer.

[0026] In some embodiments, the formaldehyde concentration is 50-150 mM (e.g., 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM). In some embodiments, the formaldehyde concentration is 80-120 mM (e.g., 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, or 120 mM). In some embodiments, the formaldehyde concentration is 100 mM.

[0027] In some embodiments, the concentration of thiamine pyrophosphate is 0.5-1.5 mM (e.g., 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM). In some embodiments, the concentration of thiamine pyrophosphate is 0.8-1.2 mM (e.g., 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM, 1.0 mM, 1.05 mM, 1.1 mM, 1.15 mM, or 1.2 mM). In some embodiments, the concentration of thiamine pyrophosphate is 1 mM.

[0028] In some embodiments, the Mg 2+ The concentration is 2.5-7.5 mM (e.g., 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, or 7.5 mM). In some embodiments, the Mg... 2+ The concentration is 4-6 mM (e.g., 4.0 mM, 4.2 mM, 4.4 mM, 4.6 mM, 4.8 mM, 5.0 mM, 5.2 mM, 5.4 mM, 5.6 mM, 5.8 mM, or 6.0 mM). In some embodiments, the Mg...2+ The concentration was 5 mM.

[0029] In some embodiments, the Mg 2+ It is provided in the form of MgSO4 or MgCl2.

[0030] In some embodiments, the concentration of the buffer solution is 10-100 mM (e.g., 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM). In some embodiments, the concentration of the buffer solution is 40-60 mM (e.g., 40 mM, 42 mM, 44 mM, 46 mM, 48 mM, 50 mM, 52 mM, 54 mM, 56 mM, 58 mM, or 60 mM).

[0031] In some embodiments, the phosphate buffer is a 50 mM dipotassium hydrogen phosphate buffer at pH 7.5.

[0032] The fifth aspect of this invention provides the use of the formaldehyde enzyme mutant of the first aspect, the biomaterial of the second aspect, or the composition of the fourth aspect in any of the following: (c1) Production of glycolaldehyde; (c2) Increase the yield of glycolaldehyde; (c3) Prepare downstream products by producing glycolaldehyde.

[0033] The sixth aspect of the present invention provides a method for preparing ethanolaldehyde, comprising: contacting formaldehyde with the formaldehyde enzyme mutant described in the first aspect or the cell culture described in the second aspect under suitable catalytic conditions to catalyze the condensation of formaldehyde into ethanolaldehyde.

[0034] In some embodiments, the catalytic reaction system contains Mg 2+ And thiamine pyrophosphate.

[0035] In some embodiments, the catalytic reaction system also includes a buffer system with a pH of 7.0-8.0 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0).

[0036] In some embodiments, the buffer system is selected from phosphate buffer, HEPES buffer, or Tris-HCl buffer.

[0037] In some embodiments, the formaldehyde concentration in the catalytic reaction system is 50-150 mM (e.g., 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM). In some embodiments, the formaldehyde concentration in the catalytic reaction system is 80-120 mM (e.g., 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, or 120 mM). In some embodiments, the formaldehyde concentration in the catalytic reaction system is 100 mM.

[0038] In some embodiments, the concentration of thiamine pyrophosphate in the catalytic reaction system is 0.5-1.5 mM (e.g., 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM). In some embodiments, the concentration of thiamine pyrophosphate in the catalytic reaction system is 0.8-1.2 mM (e.g., 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM, 1.0 mM, 1.05 mM, 1.1 mM, 1.15 mM, or 1.2 mM). In some embodiments, the concentration of thiamine pyrophosphate in the catalytic reaction system is 1 mM.

[0039] In some embodiments, Mg in the catalytic reaction system 2+ The concentration is 2.5-7.5 mM (e.g., 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, or 7.5 mM). In some embodiments, Mg in the catalytic reaction system 2+ The concentration is 4-6 mM (e.g., 4.0 mM, 4.2 mM, 4.4 mM, 4.6 mM, 4.8 mM, 5.0 mM, 5.2 mM, 5.4 mM, 5.6 mM, 5.8 mM, or 6.0 mM). In some embodiments, Mg in the catalytic reaction system... 2+ The concentration was 5 mM.

[0040] In some embodiments, the Mg 2+ It is provided in the form of MgSO4 or MgCl2.

[0041] In some embodiments, the concentration of the buffer solution is 10-100 mM (e.g., 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM). In some embodiments, the concentration of the buffer solution is 40-60 mM (e.g., 40 mM, 42 mM, 44 mM, 46 mM, 48 mM, 50 mM, 52 mM, 54 mM, 56 mM, 58 mM, or 60 mM).

[0042] In some embodiments, the phosphate buffer is a 50 mM dipotassium hydrogen phosphate buffer at pH 7.5.

[0043] The seventh aspect of the present invention provides a method for preparing glycolaldehyde, comprising: fermenting and culturing the recombinant microorganisms described in the second aspect to obtain glycolaldehyde from the fermentation broth, wherein the fermentation culture medium contains formaldehyde.

[0044] The beneficial effects of this invention are: The formaldehyde enzyme mutant provided by this invention can regulate the ratio of two-carbon and three-carbon products generated by the condensation of formaldehyde. Compared with formaldehyde enzyme, it has improved activity in catalyzing the condensation of formaldehyde into glycolaldehyde and reduced activity in catalyzing the condensation of formaldehyde into 1,3-dihydroxyacetone. Attached Figure Description

[0045] Figure 1 The relative activities of formaldehyde enzyme and mutants in the production of GA and DHA are shown. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0047] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0048] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0049] In this document, the term "mutation" in relation to amino acid positions or residues refers to the substitution of an amino acid at a specific position by another amino acid. The form "XaY" is used to denote amino acid mutations or substitutions, where 'a' represents the position of the amino acid in SEQ ID No: 2, 'X' represents the wild-type amino acid at position 'a' in SEQ ID No: 2, and 'Y' represents the amino acid resulting from the mutation at position 'a' in SEQ ID No: 2. For example, "I28L" indicates that, compared to SEQ ID No: 2, isoleucine I at position 28 of SEQ ID No: 2 is replaced by leucine L.

[0050] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to refer to a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding a formaldehyde enzyme mutant of the present invention. The term "expression cassette" of the present invention refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a formaldehyde enzyme mutant of the present invention.

[0051] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the formaldehyde enzyme mutant of this invention. Host cells include cultured cells, such as cultured mammalian cells (e.g., CHO cells, HEK293 cells, BHK cells), yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0052] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0053] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0054] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0055] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0056] The sequence information involved in the embodiments of the present invention is shown in the following table: Table 1: Sequence Information

[0057] Example 1 Construction of formaldehyde recombinant plasmid In this embodiment, formaldehyde lyase wFLS was obtained by mutation of benzaldehyde lyase PfFLS. Benzaldehyde lyase PfFLS was used as a negative control for subsequent determination of formaldehyde catalytic activity, and formaldehyde lyase wFLS was used as a positive control. The construction methods of the recombinant plasmids of formaldehyde lyase wFLS and benzaldehyde lyase PfFLS are as follows: The protein sequence of benzaldehyde lyase PfFLS (Uniprot sequence number: Q9F4L3) was obtained, and the amino acid sequence is shown in SEQ ID NO:1. The coding gene sequence was synthesized by Suzhou Genewiz Co., Ltd., and 5' (NdeI) and 3' (BamHI) restriction endonuclease sites were added to both ends of the gene. The synthesized gene was cloned into the pET16b (Ampicillin) vector by digestion of 5' NdeI and 3' BamHI to obtain the recombinant plasmid pET16b-PfFLS.

[0058] Formaldehyde lyase wFLS was synthesized by mutating seven sites—A28I, W89R, L90T, R188H, A394G, G419N, and A480W—on benzaldehyde lyase PfFLS. The amino acid sequence of formaldehyde lyase wFLS is shown in SEQ ID NO:2. The entire gene was synthesized by Suzhou Genewise Co., Ltd., and 5' (NdeI) and 3' (BamHI) restriction endonuclease sites were added to both ends of the gene. The synthesized gene was cloned into the pET16b (Ampicillin) vector by digestion with 5' NdeI and 3' BamHI to obtain the recombinant plasmid pET16b-wFLS.

[0059] Example 2 Construction of recombinant plasmid of formaldehyde enzyme mutant Mutations were selected and combined at five sites (I28, P43, T90, L112, and N283) in the wFLS sequence (amino acid sequence shown in SEQ ID NO:2). The resulting mutants are: Mutant M1, with I28D, T90L and N283H mutations, has the amino acid sequence shown in SEQ ID NO:3; The mutant M2 has the I28L, T90L, L112T and N283H mutations, and its amino acid sequence is shown in SEQ ID NO:4; The mutant M3 has the I28L, P43S, T90L, L112T and N283H mutations, and its amino acid sequence is shown in SEQ ID NO:5.

[0060] Using site-directed mutagenesis and conventional overlap PCR technology, two pairs of overlapping primers containing mutation sites (shown in SEQ ID NO:6-13) were designed to amplify the entire plasmid using pET16b-wFLS as a template. The amplified PCR product was then digested with DpnI enzyme to remove the plasmid template from the original PCR system, resulting in recombinant plasmids pET16b-M1, pET16b-M2, and pET16b-M3 expressing the mutants.

[0061] Example 3: Construction and expression of formaldehyde enzyme and mutant strains Construction of a functional recombinant strain: To facilitate subsequent enzyme activity assays, a specific strain, BL21(DE3)△frmRAB (preparation method see Dong, X., Sun, C., Guo, J., Ma, X., Xian, M., & Zhang, R. (2023)), was selected. Highly efficient biosynthesis of 2,4-dihydroxybutyric acid by a methanol assimilation pathway in engineered... Escherichia coli . Green Chemistry , 25 (19), 7662–7672. https: / / doi.org / 10.1039 / d3gc02083e) was used as the host strain for expressing formaldehyde enzyme and its mutants, as well as benzaldehyde lyase PfFLS. BL21(DE3) was purchased from Qingke Biotechnology (catalog number TSC-E01). The recombinant plasmids pET16b-wFLS, pET16b-M1, pET16b-M2, pET16b-M3, and pET16b-PfFLS constructed in Examples 1 and 2 were transformed into the strain by chemical transformation. Positive recombinant bacteria were obtained after resistance screening, and sequencing confirmed successful mutation. The constructed recombinant strains are shown in Table 1. The obtained recombinant strains were cultured to obtain formaldehyde enzyme wFLS and its mutants M1, M2, M3, and benzaldehyde lyase PfFLS.

[0062] The specific steps of the chemical conversion method are as follows: 1. Preparation of competent cells by chemical transformation: 1) Pick a single colony and incubate overnight at 37°C and 250 rpm for 12 h. 2) The next day, transfer 0.5 ml of the cells to 50 ml of antibiotic-free LB medium at 1% incubation. Incubate at 37°C and 250 rpm for approximately 2 h until OD600 = 0.4-0.6. After thorough ice bath, incubate at 4°C and 4000×g for 5 min. 3) Gently resuspend the cells in 1 ml of pre-chilled 0.1M CaCl2, bring the volume to 20 ml, and incubate on ice for 30 min. 4) Discard the supernatant and add 1.4 ml of 0.1M CaCl2 and 0.6 ml of 50% sterile glycerol. 5) Aliquot into 100 μl containers and freeze at -80°C.

[0063] 2. Mix the recombinant plasmid (plasmid concentration 50 ng / μl) with competent cells on ice at a volume ratio of 1:30, incubate on ice for 30 min, heat shock at 42℃ for 1 min, then incubate on ice for 2 min, add LB liquid medium, revive at 37℃ for 50 min, then spread on LB solid medium with the corresponding resistance, and incubate upside down at 37℃ for 12 h.

[0064] The culture conditions for the recombinant strain are as follows: (1) Seed culture method: add ampicillin (100 μg / mL) to LB medium according to the resistance of the transformed plasmid, inoculate single colonies into 2 mL LB medium in a shake tube, and culture overnight at 37°C and 200 rpm.

[0065] (2) Scale-up culture and expression: Transfer 0.5 mL of bacterial culture to 50 mL of resistant LB medium and incubate at 37 °C and 200 rpm for about 3 h until OD reaches 0.5 mL. 600 The concentration was 0.6-0.8. The shaker was cooled to 18°C, and after the bacterial culture cooled, 0.25 mM IPTG was added. Expression was induced overnight at 18°C ​​and 200 rpm for 16-20 h. The cells were then collected by centrifugation at 4000 rpm for 30 min at 4°C. Whole-cell assays were subsequently performed.

[0066] The LB liquid culture medium (1L) used in this embodiment of the invention contains 10g peptone, 10g sodium chloride, and 5g yeast extract; the LB solid culture medium (1L) contains 10g peptone, 10g sodium chloride, 5g yeast extract, and 15g agar powder.

[0067] Table 1: Nomenclature of formaldehyde enzymes and mutants, vector plasmids, strains and resistance

[0068] Example 4: Activity determination of catalytic formaldehyde to formaldehyde GA and DHA The activity of PfFLS, wFLS, M1, M2, and M3 in catalyzing the conversion of formaldehyde to glycolaldehyde (GA) and 1,3-dihydroxyacetone (DHA) was determined using a whole-cell catalysis method. The reaction system is as follows: OD 600 =30 whole cells expressing the corresponding protein (i.e., the bacterial cells collected in Example 3), 50 mM pH 7.5 dipotassium hydrogen phosphate buffer, 5 mM magnesium chloride, 1 mM thiamine pyrophosphate, 100 mM formaldehyde. The reaction was carried out at 30°C for 24 hours. Immediately after the reaction, the cells were centrifuged at 10,000 rpm for 1 min. The supernatant was filtered through a 0.22 μM filter membrane, and the products GA and DHA were detected by HPLC. The results were analyzed to quantify the activity of formaldehyde enzyme and the mutant in catalyzing the production of GA and DHA from formaldehyde based on the amount of product generated.

[0069] Determination of GA and DHA products: After the reaction, centrifuge immediately to remove cells containing formaldehyde enzyme, collect the supernatant, filter through a 0.22 μm filter membrane, and perform high-performance liquid chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad, 300 nm). HPLC analysis was performed on a column with a diameter of 7.8 mm. The HPLC settings were as follows: mobile phase: 5 mM H2SO4; injection volume: 2 μl; flow rate: 0.6 mL / min; column temperature: 15 °C; detection was performed using a RID differential detector (35 °C).

[0070] The activity of wFLS was defined as 1, and the others were relative activities compared to wFLS. The results are as follows: Figure 1 As shown, both M1 and M2 exhibited stronger activity than wFLS in catalyzing the condensation of formaldehyde into the two-carbon product ethanolaldehyde, while their activity in producing the three-carbon product 1,3-dihydroxyacetone decreased. Meanwhile, M3 only produced the two-carbon product ethanolaldehyde, and its activity in producing the two-carbon product was nearly three times higher than that of wFLS.

[0071] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A formaldehyde enzyme mutant comprising an amino acid sequence having at least 95% identity with the amino acid sequence of FLS, the amino acid sequence of which is shown in SEQ ID NO:2; The formaldehyde enzyme mutant contains mutations at at least three sites: I28, T90, and N283. The formaldehyde enzyme mutant contains at least aspartic acid or leucine at position 28, leucine at position 90, and histidine at position 283.

2. The formaldehyde enzyme mutant according to claim 1, characterized in that, The formaldehyde enzyme mutant also includes mutations at the P43 and / or L112 sites; Preferably, the formaldehyde enzyme mutant further includes serine at position 43 and / or threonine at position 112; Preferably, the formaldehyde enzyme mutant contains amino acid substitutions for I28D, T90L, and N283H; or, The formaldehyde enzyme mutant contains amino acid substitutions of I28L, T90L, L112T, and N283H; or, The formaldehyde enzyme mutant contains amino acid substitutions of I28L, P43S, T90L, L112T, and N283H. Preferably, the formaldehyde enzyme mutant comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; Preferably, the amino acid sequence of the formaldehyde enzyme mutant is as shown in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:

5.

3. Biomaterials related to formaldehyde enzyme mutants, wherein the biomaterials comprise any one of (a1)-(a5): (a1) A nucleic acid molecule encoding a formaldehyde enzyme mutant according to claim 1 or 2; (a2) An expression vector comprising the nucleic acid molecule described in (a1); (a3) A host cell containing the nucleic acid molecule described in (a1) or the expression vector described in (a2); (a4) A cell culture comprising the host cells cultured under conditions suitable for the expression of the formaldehyde enzyme mutant in the host cells described in (a3); (a5) A recombinant microorganism comprising the nucleic acid molecule of (a1) or the expression vector of (a2), wherein the recombinant microorganism is an endogenous microorganism containing an operon encoding a formaldehyde detoxification system and wherein such operon is missing or inactivated.

4. The biomaterial according to claim 3, characterized in that, The microorganism is Escherichia coli, specifically Escherichia coli with a missing or inactivated formaldehyde detoxification operon.

5. A method for producing a formaldehyde enzyme mutant, comprising: The recombinant host cells are cultured in a culture medium under conditions suitable for the host cells expressing the formaldehyde enzyme mutant as described in claim 3. And the formaldehyde enzyme mutant was obtained from cell culture.

6. A composition comprising: (b1) the formaldehyde enzyme mutant of claim 1 or 2 or the cell culture of claim 3; and (b2) Formaldehyde, thiamine pyrophosphate, Mg 2+ .

7. The composition according to claim 6, characterized in that, The composition also includes a buffer system with a pH of 7.0-8.0; Preferably, the buffer system is selected from phosphate buffer, HEPES buffer, or Tris-HCl buffer; Preferably, the concentration of formaldehyde is 50-150 mM; more preferably, it is 100 mM. Preferably, the concentration of thiamine pyrophosphate is 0.5-1.5 mM; more preferably, it is 1 mM. Preferably, the Mg 2+ The concentration is 2.5-7.5 mM; preferably 5 mM; Preferably, the phosphate buffer is a 50mM dipotassium hydrogen phosphate buffer at pH 7.

5.

8. Use of the formaldehyde enzyme mutant of claim 1 or 2, the biomaterial of claim 3, or the composition of claim 6 or 7 in any of the following: (c1) Production of glycolaldehyde; (c2) Increase the yield of glycolaldehyde; (c3) Prepare downstream products by producing glycolaldehyde.

9. A method for preparing glycolaldehyde, comprising: Under suitable catalytic conditions, formaldehyde is contacted with the formaldehyde enzyme mutant of claim 1 or 2 or the cell culture of claim 3 to catalyze the condensation of formaldehyde into glycolaldehyde. Preferably, the catalytic reaction system contains Mg 2+ and thiamine pyrophosphate; Preferably, the catalytic reaction system further includes a buffer system with a pH value of 7.0-8.

0.

10. A method for preparing glycolaldehyde, comprising: The recombinant microorganism described in claim 3 or 4 is fermented to obtain glycolaldehyde from the fermentation broth, wherein the fermentation medium contains formaldehyde.

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