Property-changed dihydroxyacetone kinase mutant and application thereof

Abstract

The invention provides a property-changed dihydroxyacetone kinase mutant and application thereof, and relates to the technical field of enzyme engineering. Through rational design and molecular modification, wild dihydroxyacetone kinase is subjected to single-point mutation and combined mutation, a series of enzyme mutants with enhanced thermal stability are obtained, and the catalytic activity of the mutants under the high-temperature condition is remarkably superior to that of wild enzymes. The invention further provides a multienzyme coupling reaction system containing the dihydroxyacetone kinase mutant, and the multienzyme coupling reaction system is used for efficiently catalyzing the process of synthesizing lactic acid by taking dihydroxyacetone as a substrate. The thermostable dihydroxyacetone kinase mutant provided by the invention solves the key bottleneck that a wild enzyme is easy to thermally inactivate under industrial application conditions, and has important industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a modified dihydroxyacetone kinase mutant and its applications. Background Technology

[0002] Dihydroxyacetone kinase (DAK, EC 2.7.1.29), a key enzyme in the microbial glycerol metabolism pathway, primarily functions to catalyze the phosphorylation of dihydroxyacetone (DHA) to generate the central metabolic intermediate dihydroxyacetone phosphate (DHAP). The specific reaction formula is as follows: DHA + ATP → DHAP + ADP

[0003] This enzyme has significant application value in the field of biotechnology. On the one hand, it plays a key role in the high-value conversion of glycerol—in microbial cell factories using inexpensive glycerol as a carbon source, DAK is an important entry point for glycerol into the central metabolism, and its activity directly determines the synthesis efficiency of chemicals such as 1,3-propanediol and butanediol. On the other hand, it is also a core component of in vitro multi-enzyme catalytic systems and can be used to construct ATP regeneration cycles and C3 chemical unit (DHAP) supply modules. For example, when used in conjunction with aldolase, it can synthesize important chemical building blocks such as chiral alcohols from DHA and various aldehyde substrates.

[0004] However, naturally occurring DAKs derived from thermophilic bacteria generally suffer from poor thermal stability, rapidly inactivating at industrially relevant temperatures of 40-50°C. This leads to a series of process challenges: reactions must be carried out at lower temperatures, resulting in limited reaction rates and low overall efficiency; in reactors requiring long-term operation or repeated enzyme use, the short enzyme lifespan causes poor process stability; and low-temperature environments also increase the risk of microbial contamination. Although screening for naturally occurring thermostable DAKs from exothermic bacteria is a potential approach, these enzymes often face challenges such as difficulty in expression, low specific activity, or insufficient compatibility with existing industrial systems, limiting their practical application. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention employs rational design and molecular modification to perform site-directed mutagenesis on wild-type dihydroxyacetone kinase, resulting in a series of enzyme mutants with enhanced thermal stability. These mutants exhibit significantly better structural stability and catalytic activity retention under high-temperature conditions compared to the wild-type enzyme.

[0006] The first object of the present invention is to provide a mutant of dihydroxyacetone kinase, said mutant having undergone any of the following mutations in the amino acid sequence shown in SEQ ID NO. 1 as the parent sequence: The alanine at position 123 is mutated to lysine; The glycine at position 326 is mutated to alanine; The serine at position 362 is mutated to aspartic acid; The glutamic acid at position 380 is mutated to asparagine; The glycine at position 381 is mutated to aspartic acid; The alanine at position 395 is mutated to valine; The serine at position 482 is mutated to threonine; The threonine at position 494 is mutated to aspartic acid; The glutamic acid at position 559 is mutated to lysine; The alanine at position 569 is mutated to serine.

[0007] Furthermore, the mutant undergoes any of the following mutations with the amino acid sequence shown in SEQ ID NO.1 as the parent sequence: The serine at position 362 is mutated to aspartic acid, and the alanine at position 18 is mutated to lysine. The serine at position 362 is mutated to aspartic acid, the alanine at position 18 is mutated to lysine, and the glycine at position 193 is mutated to alanine. The serine at position 362 is mutated to aspartic acid, the alanine at position 18 is mutated to lysine, and the glycine at position 326 is mutated to alanine. The serine at position 362 is mutated to aspartic acid, the alanine at position 18 is mutated to lysine, the glycine at position 326 is mutated to alanine, and the alanine at position 123 is mutated to leucine. The serine at position 362 is mutated to aspartic acid, and the alanine at position 123 is mutated to glutamine. The serine at position 362 is mutated to aspartic acid, the glycine at position 326 is mutated to alanine, and the serine at position 323 is mutated to threonine.

[0008] Furthermore, the amino acid sequence of the mutant is shown in any one of SEQ ID NO.2-17.

[0009] Furthermore, the mutant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the above-mentioned amino acid sequence, and has dihydroxyacetone kinase activity.

[0010] A second objective of this invention is to provide a gene encoding the aforementioned mutant.

[0011] A third objective of this invention is to provide an expression vector containing the aforementioned genes.

[0012] A fourth objective of the present invention is to provide a host cell comprising the above-described expression vector.

[0013] Furthermore, the host cell is a non-plant cell.

[0014] Furthermore, the host cell is Escherichia coli.

[0015] A fifth objective of this invention is to provide the use of the above-mentioned mutant, the above-mentioned gene, the above-mentioned expression vector, or the above-mentioned host cell in the production of lactic acid.

[0016] A sixth objective of this invention is to provide a method for producing lactic acid using dihydroxyacetone as a substrate, comprising the step of adding the aforementioned mutant to a reaction system for reaction, wherein the reaction system further comprises dihydroxyacetone, pyruvate dehydrogenase, and lactate dehydrogenase.

[0017] Furthermore, the reaction system also contains sodium ions and magnesium ions.

[0018] Furthermore, the reaction temperature is 30-60℃.

[0019] The beneficial effects of this invention are: This invention, through rational modification of key sites of dihydroxyacetone kinase, yields a mutant that exhibits significantly higher residual activity than the wild type after incubation at high temperatures (50-60°C), demonstrating excellent heat resistance. This enhanced thermal stability allows the mutant to catalyze reactions at higher temperatures, which not only accelerates the reaction rate and improves production efficiency but also reduces the risk of microbial contamination and increases substrate solubility, thereby significantly optimizing the production process and reducing production costs. This invention proposes a multi-enzyme catalytic system centered on this heat-stable dihydroxyacetone kinase, which can be used for the efficient and environmentally friendly conversion of dihydroxyacetone into platform chemicals such as lactic acid. This system exhibits good stability and high conversion efficiency, providing a stable and reliable enzyme catalyst solution for the continuous industrial production of related products. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0021] The amino acid sequence of the wild-type dihydroxyacetone kinase (WT) involved in this invention is shown in SEQ ID NO.1, specifically as follows: MSSKHWDYKKDLVLSHLAGLCQSNPHVRLIESERVVISAENQEDKITLISGGGSGHEPLHAGFVTKDGLLDAAVAGFIFASPSTKQIFSAIKAKPSKKGTLIIVKNYTGDILHFGLAAEKAKAEGLNAELLIVQDDVSVGKAKNGLVGRRGL AGTSLVHKILGAKAYLQKDNLELHQLVTFGEKVVANLVTIGASLDHVTIPARANKQEEDDSDDEHGYEVLKHDEFEIGMGIHNEPGIKKSSPIPTVDELVAELLEYLLSTTDKDRNYVQFDKNDEVVLLINNLGGTSVLELYAIQNIVVDQL ASKYSIKPVRIFTGTFTTSLDGPGFSITLLNATKTGDKDILKFLDHKTSAPGWNSNISDWSGRVDNFIVAAPEIDEGDSSSKVSVDAKLYADLLESGVKKVISKEPKITLYDTVAGDGDCGETLANGSNAILKALAEGKLDLKDGVKSLVQI TDIVETAMGGTSGGLYSIFISALAKSLKEKELSEGAYTLTLETISGSLQAALQSLFKYTRARTGDRTLIDALEFVKEFAKSKDLKLANKAAHDGAEATRKLEAKFGRASYVAEEEFKQFESEGGLPDPGAIGLAALISGITDAYFKSETKL The method for determining DAK activity is as follows: The enzyme activity of dihydroxyacetone kinase (DAK) was determined using a coupled reaction system of pyruvate kinase (PK) and lactate dehydrogenase (LDH). The standard reaction system consisted of: 100 mM HEPES buffer (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 3 mM phosphoenolpyruvate (PEP), 1.5 mM NADH, 2 mM ATP, 5 mM dihydroxyacetone (DHA), and excess PK and LDH. The principle of the assay was as follows: DAK catalyzed the reaction of DHA and ATP to produce ADP; ADP was then consumed by the PK and LDH coupled system: PK utilized ADP and PEP to generate pyruvate, and LDH further catalyzed the reduction of pyruvate and oxidation of NADH; the rate of decrease in the characteristic absorbance of NADH at 340 nm was monitored in real-time using a spectrophotometer, and this rate directly reflected the catalytic activity of DAK. One unit of enzyme activity (U) was defined as the amount of enzyme required to consume 1 μmol of NADH per minute under the above conditions.

[0022] The primers (5'-3') involved in the following examples are as follows: A123K-F: CCGAAAAGGCCAAGAAGGAAGGTCTCAACGCAGAACTG; A123K-R: CTTGGCCTTTTCGGCGGCAAGACCG; A123L-F: CCGAAAAGGCCAAGCTCGAAGGTCTCAACGCAGAACTG; A123L-R: CTTGGCCTTTTCGGCGGCAAG; G326A-F: CACCACATCACTGGATGCTCCCGGTTTTTCTATCACTTTATTG; G326-R: ATCCAGTGATGTGGTGAAAGTGCCAG; S362D-F: AGACCAGTCATCTATGTTGGAGTTCCAGC; SS62D-R: TCCAACATAGATGACTGGTCTGGCAGG; E380N-F: CCCCAGAAATAGACAATGGAGACAGTTCATCTAAGGTATC; E380-R: GTCTATTTCTGGGGCAGCCAC; G381D-F: TGAACTGTCATCTTCGTCTATTTCTGGGG; G381D-R: ATAGACGAAGATGACAGTTCATCTAAGGT; A395V-F: GATGCTAAGCTGTATGTTGACCTTCTGGAGTCTGGTG; A395-R: ATACAGCTTAGCATCAACAGATACCTTAG; S482T-F: CGCTTTAGCAAAGACATTGAAAGAGAAGGAACTTTCCGAG; S482-R: CTTTGCTAAAGCGCTGATGAAAATGG; T494D-F: AAGGGTCAGATCGTAGGCACCCTCGGAAA; T494D-R: GGTGCCTACGATCTGACCCTTGAAACGAT; E559K-F:AAACTTGGCTTTGAGCTTTCTTGTGGCTT; E559K-R:AGAAAGCTCAAAGCCAAGTTTGGAAGAGC; A395V-F:GATGCTAAGCTGTATGTTGACCTTCTGGAGTCTGGTG; A395-R:ATACAGCTTAGCATCAACAGATACCTTAG; S482T-F:CGCTTTAGCAAAGACATTGAAAGAGAAGGAACTTTCCGAG; S482-R:CTTTGCTAAAGCGCTGATGAAAATGG; T494D-F:AAGGGTCAGATCGTAGGCACCCTCGGAAA; T494D-R:GGTGCCTACGATCTGACCCTTGAAACGAT; E559K-F:AAACTTGGCTTTGAGCTTTCTTGTGGCTT; E559K-R:AGAAAGCTCAAAGCCAAGTTTGGAAGAGC; A569S-F:CTCTTCTTCGCTAACATAAGATGCTCTTCCA; A569S-R:TCTTATGTTAGCGAAGAAGAGTTCAAACA; A18K-F:GGTACTCAGCCATCTGAAAGGATTATGTCAGTCCAACCCCCATG; A18-R:CAGATGGCTGAGTACCAGATCTTTCTTG; G193A-F:CCAACCTGGTTACAATTGCTGCTTCCTTGGACCATGTCACC; G193-R:AATTGTAACCAGGTTGGCAACGACC; S323T-F:CTGGCACTTTCACCACAACACTGGATGGGCCCGGTTTTTCTATC; S323T-R:TGTGGTGAAAGTGCCAGTGAAAATGCG。

[0023] Example 1: Design of DAK mutants I. Structural Analysis Structural weakness analysis: Based on high-resolution 3D structures obtained through DAK (such as X-ray diffraction or cryo-electron microscopy), and combined with structural visualization tools, a systematic evaluation of their overall conformation and local features is conducted. The analysis focuses on secondary structure composition, domain boundaries, active center conformation, and surface properties to form a preliminary assessment of structural stability. Thermal flexibility analysis: Temperature factor (B factor) data are extracted from the crystal structure, and the distribution map of B factor of main chain atoms is drawn to identify residue regions with significant structural fluctuations; Molecular dynamics simulations are performed at three temperatures (300K, 340K, and 380K), and by comparing the differences in structural fluctuations (RMSF) of various parts of the protein after simulation, hot spots with relatively large structural changes due to heat are identified. Interaction identification: Examine non-covalent interactions such as hydrogen bonds, salt bridges, and hydrophobic stacking to identify regions where the interaction strength is weaker than the average value and determine the unstable structural point.

[0024] II. Mutation Strategy Electrostatic and hydrophobic optimization: Mutations are designed to optimize salt bridges (ion pairs). Surface residues at appropriate distances are mutated into salt bridge pairs to form stronger and more stable electrostatic pairings. Simultaneously, hydrophobic stacking in the core region is optimized to enhance structural strength.

[0025] Enhancing loop stability: To address the weakness of highly flexible loop regions, proline mutations are introduced. Proline's unique rigid structure restricts the conformational freedom of the main chain, fixing the peptide chain conformation, reducing local thermal motion, and thus increasing the protein's melting temperature. Mutations are also made in daunoglycine, which causes structural instability, to moderately increase structural rigidity.

[0026] Based on the above strategy, DAK single-site mutants were obtained, and DAK multi-site combined mutants were obtained by combining the predicted key amino acid sites.

[0027] Example 2: Construction of a strain containing the DAK mutant gene Primers were designed based on the wild-type and mutant sequences of DAK. The PCR product and linearized vector pET21b were mixed in a specific ratio using a DNA Assembly Cloning Kit, and transformation was performed at 50°C for 15 min under the catalysis of recombinase. The assembled plasmid was transformed into *E. coli* DH5a competent cells, plated on plates containing AMP resistance, and incubated overnight at 37°C. After incubation, single clones were picked for PCR verification. Positive clones were cultured overnight in test tubes, and the plasmid was extracted and transformed into *E. coli* BL21(DE3) competent cells. The cells were then plated on plates containing AMP resistance and incubated overnight at 37°C to obtain the protein-expressing recombinant strain.

[0028] Example 3: Expression of DAK mutants Host bacterial activation culture: The plasmid containing the DAK wild-type / mutant expression vector plasmid... E . coli BL21(DE3) was streaked onto amp-resistant LB solid medium and incubated overnight at 37°C. Single colonies were picked and inoculated into test tubes containing 5 mL of LB liquid medium. The test tubes were then placed in a rotary shaker at 200 rpm and incubated at 37°C for 16 h.

[0029] Fermentation culture: Inoculate 5 mL of the activated bacterial culture into a 2 L Erlenmeyer flask containing 1 L LB liquid medium with AMP resistance, and place it in a rotary shaker at 200 r / min and incubate at 37°C until OD. 600 The concentration should be between 0.8 and 1.0. After lowering the culture temperature to 16°C, add IPTG to a final concentration of 0.5 mM and continue induction under these conditions for 12-18 hours.

[0030] Separation and Purification: After overnight culture, centrifuge to remove the supernatant. Collect the precipitate in a 50 mL centrifuge tube for protein purification. Resuspend the precipitate in 30 mL of 25 mM Tris-HCl and 500 mM NaCl buffer, and vortex to mix. Add 40 mL of Tris buffer lysis buffer to the bacterial suspension, mix, and autoclave. Centrifuge at 4 °C, remove the precipitate, and transfer the supernatant to a pre-treated nickel column for binding. Wash with 100 mL of 25 mM Tris-HCl, 500 mM NaCl, and 25 mM imidazole buffer, and then elute the protein with 15 mL of 25 mM Tris-HCl (500 mM NaCl, 250 mM imidazole) buffer. Dialyze the 15 mL protein eluent overnight in 25 mM Tris-HCl and 500 mM NaCl buffer. Concentrate the 15 mL protein eluent through an ultrafiltration tube at 3500 tpm and 4 °C to 0.5–1 mL. The Bradford method was used to determine protein concentration, ensuring that all mutant proteins had uniform concentrations before detection.

[0031] Example 4: Determination of enzyme activity and thermostability of DAK and its mutants A pyruvate kinase (PK) and lactate dehydrogenase (LDH) coupled reaction system was used. After the reaction was initiated, the enzyme activity of purified DAK wild-type and mutants was determined by spectrophotometry at 340 nm at 30℃, monitoring NADH consumption in real time. The purified DAK wild-type and mutants were incubated at 50℃, 55℃, or 60℃ for 30 minutes and then cooled in an ice bath. Residual enzyme activity was then measured using the PK-LDH coupled system (calculated with the enzyme activity of wild-type WT at 30℃ as 100%). Single mutants or combined mutants of DAK with improved thermostability compared to wild-type were screened, as shown in Table 1.

[0032] Table 1 Enzyme activity and thermostability of DAK and its mutants

[0033] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A mutant of dihydroxyacetone kinase, characterized in that, The mutant undergoes any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1, as in the parent sequence: The alanine at position 123 is mutated to lysine; The glycine at position 326 is mutated to alanine; The serine at position 362 is mutated to aspartic acid; The glutamic acid at position 380 is mutated to asparagine; The glycine at position 381 is mutated to aspartic acid; The alanine at position 395 is mutated to valine; The serine at position 482 is mutated to threonine; The threonine at position 494 is mutated to aspartic acid; The glutamic acid at position 559 is mutated to lysine; The alanine at position 569 is mutated to serine.

2. The mutant according to claim 1, wherein The mutant undergoes any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1, as in the parent sequence: The serine at position 362 is mutated to aspartic acid, and the alanine at position 18 is mutated to lysine. The serine at position 362 is mutated to aspartic acid, the alanine at position 18 is mutated to lysine, and the glycine at position 193 is mutated to alanine. The serine at position 362 is mutated to aspartic acid, the alanine at position 18 is mutated to lysine, and the glycine at position 326 is mutated to alanine. The serine at position 362 is mutated to aspartic acid, the alanine at position 18 is mutated to lysine, the glycine at position 326 is mutated to alanine, and the alanine at position 123 is mutated to leucine. The serine at position 362 is mutated to aspartic acid, and the alanine at position 123 is mutated to glutamine. The serine at position 362 is mutated to aspartic acid, the glycine at position 326 is mutated to alanine, and the serine at position 323 is mutated to threonine.

3. The mutant according to claim 1 or 2, characterized in that: The amino acid sequence of the mutant is shown in any one of SEQ ID NO. 2-17.

4. The gene encoding any of the mutants described in claims 1-3.

5. An expression vector comprising the gene of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. The use of any of the mutants described in claims 1-3, the gene described in claim 4, the expression vector described in claim 5, or the host cell described in claim 6 in the production of lactic acid.

8. A method for producing lactic acid using dihydroxyacetone as a substrate, characterized in that: The step includes adding the mutants described in claims 1-3 to the reaction system.

9. The method of claim 8, wherein: The reaction system also contains dihydroxyacetone, pyruvate dehydrogenase, and lactate dehydrogenase.

10. The method of claim 8, wherein: The reaction system also contains sodium ions and magnesium ions.

Images