An aldolase mutant and its use in producing 3-hydroxypropionaldehyde

Abstract

The application discloses an aldolase mutant and application thereof in production of 3-hydroxypropionaldehyde, and relates to the technical field of enzymes. The application provides an aldolase mutant, and the amino acid sequence is shown as SEQ ID NO:1. The aldolase mutant constructed by the application has an enzyme activity which is 7768% higher than that of a wild type, and the yield of 3-hydroxypropionaldehyde produced by the aldolase is improved. yqhD On the basis of the mutant, the expression host is subjected to gene knockout, the conversion rate of formaldehyde is improved to 96.3%, and the conversion rate of acetaldehyde is improved to 58.94%. The mutant has a strong formaldehyde tolerance, can tolerate a formaldehyde concentration of up to 475mM, and has an industrial strain level tolerance, solves the core bottleneck of inhibiting enzyme activity in the process of biosynthesis of 3-hydroxypropionaldehyde, and has a wide application prospect in an industrial production scene.

Description

Technical Field

[0001] This invention relates to the field of enzyme technology, specifically to an aldolase mutant and its application in the production of 3-hydroxypropional. Background Technology

[0002] 3-Hydroxypropionaldehyde (3-HPA) is an important platform compound with high reactivity. Its molecule contains two active functional groups, hydroxyl and aldehyde groups, and can be transformed into a series of high value-added products through various reactions such as reduction, oxidation, esterification, and polymerization. It has broad application prospects in many fields such as medicine, chemical industry, and materials.

[0003] Currently, the main methods for producing 3-hydroxypropanal include chemical synthesis and biosynthesis. Traditional chemical synthesis mainly involves reaction routes such as acrolein hydration and ethylene oxide hydroformylation, but it suffers from drawbacks such as harsh reaction conditions (e.g., high temperature and high pressure), numerous byproducts, low product selectivity, and difficulties in subsequent separation and purification, making it difficult to meet the demands of modern green chemical industries for efficient, environmentally friendly, and low-energy-consumption production processes. In contrast, biosynthesis, with its significant advantages such as mild reaction conditions (room temperature and pressure), high substrate specificity, fewer byproducts, and environmental friendliness, has become a research hotspot and development direction for the synthesis of 3-hydroxypropanal.

[0004] In the biosynthetic pathway of 3-hydroxypropanal, the aldolase-catalyzed condensation reaction of formaldehyde and acetaldehyde is one of the core steps. Aldolases are enzymes that catalyze the condensation reactions of aldehyde and ketone compounds. They specifically catalyze the aldol condensation reaction of formaldehyde and acetaldehyde to produce 3-hydroxypropanal. This reaction is characterized by its wide availability of substrates and high reaction specificity, making it a key rate-limiting step in the biosynthesis of 3-hydroxypropanal. However, naturally occurring wild-type aldolases face significant technical bottlenecks in practical applications, severely limiting the industrial-scale promotion of the 3-hydroxypropanal biosynthesis process.

[0005] Existing research has revealed the concentration-dependent behavior and mechanisms of different aldolases, generally showing that aldolases retain activity at lower concentrations, but their activity decreases at higher concentrations of formaldehyde substrate. Most studies have shown optimal activity at 20-40 mM, but the production capacity at this concentration is not suitable for industrial applications.

[0006] A 2025 study published in *ACS Catalysis* by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, titled "Helix Zipper Regulating Formolase Activity," systematically determined the activity changes of the artificial formaldehyde condensate enzyme formolase and its mutants within a formaldehyde concentration range of 5–180 mM. Through engineering modification of the substrate channel helix, the K6 mutant of formolase showed a 27.3-fold increase in activity at 20 mM and an 86.5-fold increase at 40 mM. However, above 300 mM, it was irreversibly inactivated due to the cross-linking of lysine and asparagine residues with formaldehyde.

[0007] A 2021 report in *ACS Synthetic Biology* described an aldolase-based new pathway for bioconversion of formaldehyde and ethanol into 1,3-propanediol in *Escherichia coli*. DERA derived from the thermophilic bacterium *Thermotoga maritima* was introduced into *E. coli* to synthesize 1,3-propanediol. The optimal balance of the metabolic pathway was observed when the formaldehyde concentration was controlled at approximately 0.6 mM. Above this threshold, inhibition occurred; after incubation with 300 mM formaldehyde for 2 hours, enzyme activity decreased to 61%.

[0008] DERA, a class I aldolase, relies on the lysine residue in its active site for catalyzing the aldol condensation reaction. However, the ε-amino group of this lysine side chain is also the target site for the Schiff base reaction of formaldehyde. High concentrations of free formaldehyde may undergo irreversible addition with the lysine residue in the catalytic site, or lead to intermolecular cross-linking to form a Schiff base, thereby destroying enzyme activity. However, in this study, mutations in the key lysine residues K179 and K208 resulted in the complete loss of aldolase activity, suggesting that optimizing the aldolase's tolerance presents certain technical challenges.

[0009] First, wild-type aldolases exhibit low catalytic efficiency for formaldehyde and acetaldehyde, specifically a small catalytic constant Kcat and a low catalytic efficiency index Kcat / Km, resulting in a slow reaction rate. Second, formaldehyde, as one of the reaction substrates, requires a certain concentration in the reaction system to ensure reaction efficiency. However, formaldehyde itself is toxic and can disrupt the spatial conformation of the aldolase and damage its active site, leading to a significant decrease in enzyme activity or even complete inactivation. This prevents the formaldehyde concentration in the reaction system from being effectively increased, thus limiting the substrate conversion rate and the yield of 3-hydroxypropionaldehyde, failing to meet the capacity requirements for industrial production.

[0010] To address the aforementioned issues, the biosynthetic efficiency of 3-hydroxypropionaldehyde has been improved through enzyme molecule modification and host engineering optimization. Although some research has explored this approach, a mutant aldolase with significantly enhanced formaldehyde tolerance and superior catalytic efficiency has yet to be obtained. Furthermore, a technical solution combining host engineering optimization to construct highly efficient 3-hydroxypropionaldehyde synthesizing strains remains to be developed. Therefore, developing a highly efficient aldolase mutant and constructing a corresponding highly efficient engineered strain is of great significance for promoting the industrial application of 3-hydroxypropionaldehyde biosynthesis and is currently a key research focus and urgent need in this field. Summary of the Invention

[0011] The purpose of this invention is to provide an aldolase mutant that can tolerate high concentrations of formaldehyde and its application in the production of 3-hydroxypropionaldehyde.

[0012] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an aldolase mutant, the amino acid sequence of which is shown in SEQ ID NO:1.

[0013] SEQ ID NO:1: MIEYRIEEAVAKYREFYEFKPVRESAGIEDVKSAIEHTNLKPFATPDDIKKLCLEARENRFHGVMVNPCYVKLAREELEGTDVKVVTVVGFPLGANETRTKAHEAIFAVESGADEIDMVINVGM LKAKEWEYVYEDIRSVVESVKGKVVKVIIETCYLDTEEKIAACVISKLAGAHFVKTSTGFGTGGATAEDVHLMKWIVGDEMGVKASGFIRTFEDAVKMIMYGADRIGTDSGVKIVQGGEERYGG.

[0014] According to some embodiments of the present invention, the amino acid sequence of the aldolase mutant has at least 80% similarity to the sequence shown in SEQ ID NO:1.

[0015] According to some embodiments of the present invention, the amino acid sequence of the aldolase mutant has at least 85% similarity to the sequence shown in SEQ ID NO:1.

[0016] According to some embodiments of the present invention, the amino acid sequence of the aldolase mutant has at least 90% similarity to the sequence shown in SEQ ID NO:1.

[0017] According to some embodiments of the present invention, the amino acid sequence of the aldolase mutant has at least 95% similarity to the sequence shown in SEQ ID NO:1.

[0018] Secondly, the present invention provides a DNA molecule encoding the above-mentioned aldolase mutant.

[0019] Specifically, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO:2.

[0020] SEQ ID NO:2: .

[0021] Thirdly, the present invention provides a recombinant expression vector for producing 3-hydroxypropionaldehyde, wherein the recombinant expression vector carries the aforementioned DNA molecule.

[0022] Specifically, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0023] Furthermore, the eukaryotic expression vectors include, but are not limited to, any one or more of pPIC3, pPIC9, pYES2 / NT C, pFastBac, pEGFP-N1, pcDNA3.1, and pLVX.

[0024] Furthermore, the prokaryotic expression vectors include, but are not limited to, any one or more of pMD19, pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

[0025] Furthermore, the prokaryotic expression vector is pET28a.

[0026] According to some embodiments of the present invention, the method for constructing the recombinant expression vector is to introduce the gene sequence shown in SEQ ID NO:2 into the vector pET28a plasmid to obtain the recombinant plasmid.

[0027] Fourthly, the present invention provides a genetically engineered bacterium containing the above-described DNA molecule or the above-described recombinant expression vector.

[0028] Specifically, the host bacterium of the genetically engineered bacteria is Escherichia coli.

[0029] According to some embodiments of the present invention, the above-mentioned recombinant plasmid is transformed into Escherichia coli to obtain an Escherichia coli expression strain. BL21(DE3) .

[0030] According to some embodiments of the present invention, the method for preparing the genetically engineered bacteria is to knock out the host. BL21 (DE3) The yqhD gene, obtained △yqhD-BL21(DE3) The host transfers the aforementioned recombinant plasmid into it to obtain a mutant. △yqhD-BL21(DE3) Engineered bacteria.

[0031] Fifthly, the present invention provides the use of the above-mentioned aldolase mutant, the above-mentioned DNA molecule, the above-mentioned recombinant expression vector, or the above-mentioned genetically engineered bacteria in the production of 3-hydroxypropionaldehyde and other aldol condensates.

[0032] According to some embodiments of the present invention, the application is to use CRISPR / Ca9 gene editing to knock out the host. BL21 (DE3) The yqhD gene, obtained △yqhD-BL21(DE3) The host transfers a recombinant plasmid containing the aldolase mutant into it to obtain... mutant-△yqhD-BL21(DE3) Engineered bacteria.

[0033] The beneficial effects of this invention are as follows: The aldolase mutant constructed in this invention has significantly improved the tolerance to formaldehyde compared to the wild type. Under the conditions of 250 mM formaldehyde and 250 mM acetaldehyde substrate, the enzyme activity is increased by 7768% compared to the wild type, which greatly increases the yield of 3-hydroxypropionaldehyde produced by the aldolase.

[0034] The aldolase mutant showed a 159% increase in the catalytic constant Kcat for formaldehyde and an 18% increase in the catalytic efficiency index Kcat / Km for formaldehyde compared to the wild type. For acetaldehyde, the catalytic constant Kcat increased by 187% and the catalytic efficiency index Kcat / Km for acetaldehyde increased by 150%. The aldolase mutant demonstrated improved overall catalytic efficiency for both formaldehyde and acetaldehyde substrates after modification.

[0035] Based on this mutant, the yqhD gene was knocked out in the expressing host. mutant-△yqhD-BL21(DE3) Comparison mutant-BL21(DE3) Yield increased by 14%, while substrate conversion rates improved significantly, with formaldehyde conversion increasing by 28.20% and acetaldehyde conversion increasing by 30.41%, ultimately achieving formaldehyde and acetaldehyde conversion rates of 96.3% and 58.94%, respectively. mutant-△yqhD-BL21 (DE3) It has broad application prospects and high industrial value in the biosynthesis of 3-hydroxypropionaldehyde.

[0036] The aldolase mutant exhibited superior enzyme activity and high substrate tolerance at formaldehyde concentrations ranging from 100 to 1000 mM. Its metabolic or stress systems could tolerate formaldehyde up to 475 mM, and it maintained high activity within the 600–1000 mM range. Even at extremely high formaldehyde concentrations of 1000 mM, it maintained a 3-HPA yield of 146.39 mM, representing a 1124% increase in enzyme activity compared to the WT group and a 426% increase compared to the S233D+F43T group, demonstrating industrial-grade tolerance. Attached Figure Description

[0037] Figure 1 The image shows enzyme activity data for 24 point mutants.

[0038] Figure 2 The image shows enzyme activity data for 14 two-site mutants.

[0039] Figure 3 The image shows enzyme activity data for six three-point mutants.

[0040] Figure 4SDS-PAGE electrophoresis was used to analyze the expression of the target protein in wild-type and mutant cells; -1 is whole cells before induction, -2 is whole cells after induction, -3 is supernatant after sonication, and -4 is supernatant after heating -3 at 70℃ for 20 min.

[0041] Figure 5 For WT, mutant-BL21(DE3) and mutant-△yqhD-BL21(DE3) 3-hydroxypropanal production.

[0042] Figure 6 for mutant Compared with the control group, enzyme activity was compared at low substrate concentrations.

[0043] Figure 7 for mutant Compared with the control group, enzyme activity was compared at high substrate concentrations.

[0044] Figure 8 for mutant Compared with the tolerance of the control group. Detailed Implementation

[0045] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are only preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used is conventional equipment, and the equipment and materials used in each embodiment are the same.

[0046] Example 1 Screening and preparation of mutants (1) Construction of genetically engineered Escherichia coli: Based on the deoxyribose-5-phosphate aldolase (DERA) derived from Thermophyton floccosum, this invention designs and constructs mutants at multiple sites. First, the enzyme activities of 24 single-point mutants were constructed and measured, covering multiple potential active sites (including K20, A44, C65, Y70, G183, G212, R214, S233, etc.).

[0047] (2) The mutation site was designed and introduced into the vector plasmid pET-28a using SnapGene software to obtain the complete recombinant plasmid. The recombinant gene was then transformed into an Escherichia coli expression strain. BL21(DE3) In the process, genetically engineered Escherichia coli strains were obtained through screening.

[0048] (3) Select the selected single colonies and culture them overnight in 4 mL LB medium. Inoculate them at 2% in a shake flask at 37°C until the OD600 is 0.6-0.8. Add IPTG to induce protein expression to a final concentration of 0.2 mM and culture at 30°C for 15-20 h. Collect the cells by centrifugation at 12000 rpm for 1 min.

[0049] (4) Resuspend the bacterial cells in a centrifuge tube with 20 mM phosphate buffer solution (pH 7.0), and break the bacterial cells in an ice-water mixture environment using an ultrasonic cell disruptor (probe No. 2, power 130W, on 2 s / off 4 s, sonication for 3 min). Centrifuge at 12000 rpm for 1 min and collect the supernatant.

[0050] (5) Purification was performed using the heat-resistant properties of the target protein. The purification conditions were: heat shock at 70℃ for 20 min, followed by centrifugation at 12000 rpm for 1 min to collect the supernatant. The supernatant was then verified by SDS-PAGE. Figure 4 As shown, the processing conditions can yield a high-purity target protein solution, whose heat resistance properties have significant advantages in industrial applications.

[0051] (6) Enzyme activity screening was performed by adding 1 g / L of enzyme to a final concentration of 100 mM formaldehyde and 100 mM acetaldehyde, reacting for 1 h at 30°C and 500 rpm in a metal bath. For example... Figures 1-3 As shown, among these single-point mutants, some mutants exhibited significant increases in enzyme activity, with S233D, C65M, and G212F ranking in the top three. Other mutants, such as K179 and K208, showed complete loss or significant reduction in enzyme activity. Furthermore, the applicant combined the selected advantageous single-point mutations to construct 20 combined mutants, ultimately selecting one mutant, DERA. C65M / G212F / S233D The triple mutant is significantly more effective than any single-point or two-point mutant.

[0052] Example 2: Validation of kinetic parameters of aldolase mutant on formaldehyde substrate Formaldehyde concentration gradients were set at 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, and 40 mM. 2 g / L of heat-shock purified enzyme solution and 100 mM acetaldehyde were added, and the mixture was reacted in a metal bath at 35 °C for 10 min. Then, 40 µL of the reaction dilution was added to 80 µL of benzyloxyamine hydrochloride derivatizing reagent, and the reaction was allowed to proceed for another 20 min. The 3-hydroxypropionaldehyde yield was then detected by HPLC after membrane chromatography. The results are shown in Table 1. The catalytic constant Kcat of the modified aldolase mutant was increased by 159% compared to the wild type, and the catalytic efficiency index Kcat / Km was increased by 18%. After modification, the structure of the active site in the mutant was fine-tuned, making it more conducive to the reaction transformation. Although Km increased slightly, which may have slightly affected the initial binding tightness of the substrate, the overall catalytic efficiency was still improved.

[0053] Table 1. Kinetic parameters of aldolase on formaldehyde

[0054] Example 3: Validation of kinetic parameters of the aldolase mutant on the substrate acetaldehyde Acetaldehyde concentration gradients were set at 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, and 90 mM. 2 g / L of heat-shocked purified enzyme solution and 100 mM formaldehyde were added. The reaction was carried out in a metal bath at 35 °C for 10 min. Then, 40 µL of the diluted reaction solution was added to 80 µL of benzyloxyamine hydrochloride derivatizing reagent, and the reaction was carried out for another 20 min. After membrane chromatography, the yield of 3-hydroxypropionaldehyde was detected by HPLC. The results are shown in Table 2. The catalytic constant Kcat of the modified aldolase mutant was increased by 187% compared to the wild type, and the catalytic efficiency index Kcat / Km was increased by 150%. Combined with Table 1, it can be seen that the modified aldolase mutant improved the overall catalytic efficiency for both formaldehyde and acetaldehyde substrates. In industrial applications with high substrate concentrations, the reaction rate approached Vmax, dominated by Kcat, and its performance far exceeded that of the wild type, demonstrating its potential as a high-speed catalyst in industrial production.

[0055] Table 2. Kinetic parameters of aldolase on acetaldehyde

[0056] Example 4: Enzyme activity verification after host gene editing of aldolase mutant To increase the yield of 3-hydroxypropionaldehyde and reduce its conversion into other byproducts, CRISPR / Ca9 gene editing was used to knock out the host gene after product metabolic flux analysis. BL21(DE3) The yqhD gene, obtained △yqhD-BL21(DE3) The host transfers the mutant plasmid into it and obtains... mutant-△yqhD-BL21(DE3)Engineered bacteria. Induced whole cells were obtained using the culture method described in Example 2. Formaldehyde and acetaldehyde were added to a final concentration of 500 mM and 1000 mM respectively to achieve an OD200 concentration. 600 =200, reacted in a metal bath at 30℃ and 500rpm for 3h. Then the reaction solution was reacted with benzyloxyamine hydrochloride derivatizing agent for 20min, and the yield of 3-hydroxypropionaldehyde was detected by HPLC after membrane filtration.

[0057] The results are as follows Figure 5 As shown, increasing the amount of enzyme and substrate concentration involved in the reaction can significantly increase the yield of 3-hydroxypropionaldehyde in each group, but... mutant Compared with the wild type, the yield and substrate conversion rate of the group were significantly improved. The highest yield of 3-hydroxypropanal was 10 times that of the wild type. The formaldehyde conversion rate and acetaldehyde conversion rate (calculated as: conversion rate = 3-hydroxypropanal yield / substrate consumption * 100%) were increased by up to 48.85% and 46.77%, respectively. mutant-△yqhD-BL21(DE3) Comparison mutant-BL21(DE3) Yield increased by 14%, while substrate conversion rates were significantly improved, with formaldehyde conversion rate increasing by 28.20% and acetaldehyde conversion rate increasing by 30.41%. mutant-△yqhD-BL21(DE3) Not only did it successfully increase the yield of the target product, but it also significantly improved the substrate conversion rate. This modification can reduce raw material costs and significantly improve the economic feasibility of biosynthesis of 3-hydroxypropionaldehyde.

[0058] Comparative Example 1 Comparative Example 1 is the mutant S233D described in patent CN112921021A.

[0059] Comparative Example 2 Comparative Example 2 is the mutant S233D+F43T, S233D+N59Y, and S233D+H103F described in the literature “Ultrafast Conversion of CO2 into C3–C4 Diols in a Synergistic Electrochemical and AI-Assisted Biosynthesis System”.

[0060] Example 5: Enzyme activity verification of aldolase mutant and comparative sample at low substrate concentrations. The strain was inoculated into 5 mL of SB medium at a 2% inoculum and cultured overnight at 37°C. Subsequently, it was transferred to 100 mL of SB medium at a 2% inoculum and cultured until OD500 reached. 600After reaching a concentration of 0.6-0.8, 0.2 mM IPTG was added to induce gene expression. The cells were cultured at 30℃ for another 10 hours. The absorbance of the bacterial cells at 600 nm was measured. A certain amount of bacterial cells was collected, centrifuged to remove the supernatant, and resuspended in a certain amount of 20 mM phosphate buffer solution (pH=7.0). Formaldehyde and acetaldehyde were added to a final concentration of 250 mM and 250 mM respectively to bring the bacterial cell concentration to an OD value of [insert OD value here]. 600 =50, reacted in a metal bath at 30℃ and 500rpm for 1h. 40 µL of the diluted reaction solution was added to 80 µL of benzyloxyamine hydrochloride derivatizing reagent, incubated at room temperature for 20 min, and then 800 µL of methanol was added to terminate the reaction. The solution was filtered through a 0.2 µm organic (nylon) filter into a sample vial. A 5 µm C18 reverse-phase column (4.6 × 150 mm) with pure organic solvent as the stock solution was used. The mobile phase was ultrapure water (containing 0.1% TFA) and acetonitrile (containing 0.1% TFA). The injection volume was 10 µL. A gradient elution method was used. In the first 3 min, the acetonitrile content was 40%. From 3 to 8 min, the acetonitrile content increased from 40% to 70%, then rapidly reached 100%, held for 2 min, and then rapidly decreased back to 40% and held for 2 min. The total flow rate was 1 mL / min, the column temperature was 30 °C, and a UV detector with a detector temperature of 35 °C was used. The substance being measured could be observed at 215 nm based on the characteristic absorption of benzyloxy groups.

[0061] The results are as follows Figure 6 As shown, at substrate concentrations of 250 mM formaldehyde and 250 mM acetaldehyde, the wild-type aldolase almost lost its enzyme activity, with a yield of only 1.24 mM of 3-hydroxypropanal. In contrast, the mutant produced 97.57 mM of 3-hydroxypropanal, representing a 7768% increase in enzyme activity compared to the wild-type, a 1784% increase compared to Comparative Example 1 (S233D), and a 724%, 3410%, and 740% increase in enzyme activity compared to the mutants S233D+F43T, S233D+N59Y, and S233D+H103F described in Comparative Example 2, respectively.

[0062] Example 6: Enzyme activity verification of aldolase mutant and comparative sample at high substrate concentrations. After centrifuging the cultured bacterial cells and removing the supernatant, resuspend them in a certain amount of 20 mM phosphate buffer solution (pH=7.0). Then, add 500 mM formaldehyde and 1000 mM acetaldehyde to a final concentration to achieve an OD value of [missing value]. 600 =200, reacted in a metal bath at 30℃ and 500rpm for 3h, and then the yield of 3-HPA was measured.

[0063] The results are as follows Figure 7As shown, at substrate concentrations of 500 mM formaldehyde and 1000 mM acetaldehyde, the aldolase mutant can still produce 286.93 mM 3-hydroxypropanal, which is 782% higher than the wild-type enzyme activity, 1772% higher than the enzyme activity of Comparative Example 1 S233D, and 408%, 862%, and 5355% higher than the enzyme activities of the mutants S233D+F43T, S233D+N59Y, and S233D+H103F described in Comparative Example 2, respectively.

[0064] As shown in the results of Example 5, the S233D+N59Y and S233D+H103F mutants in Comparative Example 1 and Comparative Example 2 were significantly less effective than the mutants of the present invention in enhancing enzyme activity at low substrate concentrations. As shown in the results of Example 6, at high substrate concentrations (500mM formaldehyde and 1000mM acetaldehyde), the enzyme activity of Comparative Examples 1-2 was significantly inhibited and could not tolerate the high concentrations of substrate required for industrial production. In contrast, the aldolase mutant of the present invention exhibited excellent substrate tolerance and could maintain excellent enzyme activity in substrate environments of different concentrations (especially high concentrations), making it suitable for industrial production scenarios.

[0065] Example 7 mutant Comparison of tolerance with the control group After centrifuging the cultured bacterial cells and removing the supernatant, the cells were resuspended in a certain amount of 20 mM phosphate buffer solution (pH=7.0). Acetaldehyde was then added to a final concentration of 1000 mM, and formaldehyde was added at gradient concentrations of 100 mM, 225 mM, 350 mM, 475 mM, 600 mM, 725 mM, 850 mM, and 1000 mM to achieve a final bacterial cell concentration of OD0.05. 600 =200, reacted in a metal bath at 30℃ and 500rpm for 3h, and then the yield of 3-HPA was measured.

[0066] The results are as follows Figure 8As shown, the wild-type WT aldolase exhibits low formaldehyde tolerance, showing continuous inhibition with increasing formaldehyde levels, resulting in a gradual decrease in 3-HPA production. The dominant mutant S233D+F43T in Comparative Example 2 demonstrates some formaldehyde tolerance, with a peak tolerance at 225 mM. Production begins to decline at 350 mM, indicating limited detoxification ability and susceptibility to inhibition by high concentrations of formaldehyde. In contrast, the mutant of this invention exhibits high formaldehyde tolerance, with a peak tolerance at 475 mM, followed by a slow decline, suggesting that its metabolic or stress system can tolerate formaldehyde up to 475 mM. Crucially, the mutant of this invention demonstrates an extremely high formaldehyde tolerance far exceeding that of existing technologies, maintaining highly efficient catalytic activity even at ultra-high concentrations of 600-1000 mM, with 3-HPA production exceeding 140 mM. Under a full concentration gradient (100-1000 mM), the 3-HPA yield of the mutant of this invention was consistently significantly higher than that of the wild type (WT) and the existing mutant (S233D+F43T). Under extremely high formaldehyde stress of 1000 mM, the 3-HPA yield of the mutant of this invention was still as high as 146.39 mM, representing an increase of 1124% in enzyme activity compared to the WT group and 426% compared to the S233D+F43T group, fully demonstrating its industrial-grade substrate tolerance and catalytic stability.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An aldolase mutant, characterized in that, It is a C65M / G212F / S233D mutant, and the amino acid sequence of the aldolase mutant is shown in SEQ ID NO:

1.

2. A DNA molecule encoding the aldolase mutant of claim 1.

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

2.

4. A genetically engineered bacterium containing the aldolase mutant of claim 1 or the recombinant expression vector of the DNA molecule of any one of claims 2-3.

5. The genetically engineered bacterium according to claim 4, characterized in that, The method for preparing the genetically engineered bacteria is to knock out the host. BL21(DE3) The yqhD gene, obtained △yqhD-BL21(DE3) The host transfers the recombinant plasmid constructed according to claim 1 into it to obtain a mutant. △yqhD-BL21(DE3) Engineered bacteria.

6. The use of the aldolase mutant of claim 1 or the genetically engineered bacteria of the DNA molecule of any one of claims 2-3 in the production of 3-hydroxypropionaldehyde and other aldol condensates.

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