Dehydrogenase, mutant and application of dehydrogenase and mutant in biosynthesis of 2, 4-dihydroxybutyric acid

Abstract

The invention provides dehydrogenase, a mutant and application of the dehydrogenase and the mutant in biosynthesis of 2, 4-dihydroxybutyric acid. According to the invention, dehydrogenase key active residues capable of catalyzing hybrid enzyme activity of L-lactic acid and 2-keto-4-hydroxybutyric acid at the same time are modified to obtain mutants with improved activity, and the dehydrogenase and the mutants thereof can well take various alpha-keto acids or alpha-hydroxy acids such as L-lactic acid, pyruvic acid, 2-keto-4-hydroxybutyric acid and the like as substrates; the method has broad-spectrum substrate characteristics. The dehydrogenase and the mutant thereof can be further applied to a catalytic cascade system of a three-step enzyme reaction in which dehydrogenase participates, in the first step, L-lactic acid is dehydrogenated under the action of dehydrogenase to generate pyruvic acid, in the second step, pyruvic acid and formaldehyde are condensed under the action of aldolase to generate 2-ketone-4-hydroxybutyric acid, and in the third step, L-lactic acid and the mutant thereof are subjected to dehydrogenase reaction to generate 2-ketone-4-hydroxybutyric acid. And carrying out hydrogenation reduction on the 2-ketone-4-hydroxybutyric acid under the action of dehydrogenase to obtain the 2, 4-dihydroxybutyric acid. The catalytic cascade system has the advantages of ultrahigh atom economy, no waste generation, no pollution and high yield.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to novel dehydrogenases and mutants and their application in the biosynthesis of 2,4-dihydroxybutyric acid. Background Technology

[0002] Formaldehyde is an important metabolic intermediate of a one-carbon compound. One-carbon resources such as CO2, methanol, formic acid, and methane can all be converted into formaldehyde through chemical or biological methods. Therefore, formaldehyde is an ideal raw material that is inexpensive and abundant.

[0003] Lactic acid can be produced in large quantities through microbial fermentation. Due to its widespread applications in food, beverages, pharmaceuticals, and bioplastics, numerous factories have been built, significantly increasing production capacity and reducing costs. Therefore, lactic acid is an ideal, inexpensive raw material for biosynthesis.

[0004] 2,4-Dihydroxybutyric acid (DHB) can be synthesized in a one-step chemical conversion with methanethiol to form the methionine analog 2-hydroxy-4-methylthiobutyric acid (KMTB). It can also serve as a precursor for the synthesis of bulk chemicals such as 2-keto-4-methylthiobutyrate (KMTB) and γ-butyrolactone (GBL). The annual production of these chemicals ranges from tens to hundreds of thousands of tons, and demand continues to rise. DHB can also be used as a substrate in the fermentation synthesis of 1,3-propanediol, 1,2,4-butanetriol, and 1,4-butanediol. These compounds are bulk chemicals with wide applications as precursors in plastics, solvents, and aerospace propellants. Therefore, DHB is a platform molecule with high industrial value potential. However, its high price and limited availability by manufacturers significantly restrict its application. Therefore, developing inexpensive and efficient biosynthetic methods for DHB is of significant practical value.

[0005] However, DHB has not discovered any natural biosynthetic pathways. Currently, feasible non-natural biosynthetic pathways have been shown to have drawbacks such as low yields and inability to compete with chemical methods. For example, Walther et al. (Walther, T.; Calvayrac, F.; Malbert, Y.; Alkim, C.; Dressaire, C.; Cordier, H.; JM, Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine. Metabolic Engineering 2018, 45, 237-245.) reported the simplest two-step enzymatic method, which uses homoserine as a raw material, converts it to 2-keto-4-hydroxybutyric acid by transaminase, and then reduces it to DHB by dehydrogenase. This two-step enzymatic synthesis method produced only 5.3 g / L DHB after 48 hours of fermentation with E. coli. Subsequently, Liu et al. (Liu, Y.; Zhang, Jn; Li, R.; Yu, B., Efficient Production of 2,4-Dihydroxybutyrate from l-Homoserine by the Designed Cofactor Self-SufficientRoute. ACS Sustainable Chemistry & Engineering 2022, 10(43), 14361-14369.) increased the yield to 17.8 g / L (44 h) by introducing an NAD cofactor recycling system, which is the highest yield reported to date. However, this method, due to the introduction of the cofactor recycling system, resulted in the consumption of some irrelevant substrates, the introduction of enzymes, and the generation of byproducts, leading to low atom utilization and high cost. In addition, Li et al. (CN112921021A, 2023) designed a DHB biosynthesis route based on the one-carbon raw material formaldehyde methanol, which has the characteristics of high atom economy and low raw material price, but its yield is low, about 10 mM (1.2 g / L). The main reason lies in the low activity of the dehydrogenases in the pathway, particularly their poor affinity for the substrate. This makes it difficult for them to utilize the low-content intermediate substrate 2-keto-4-hydroxybutyric acid in the catalytic system, resulting in low efficiency of the entire catalytic system and consequently low yield. Therefore, obtaining dehydrogenases with high substrate affinity to achieve efficient catalysis of L-lactate dehydrogenation and hydrogenation of low-concentration 2-keto-4-hydroxybutyric acid is of great significance for increasing the yield of 2,4-dihydroxybutyric acid. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention utilizes bioinformatics methods to discover and obtain various dehydrogenases with higher activity and substrate affinity. Through mutation modification, their activity and affinity are enhanced, and their substrate preferences are altered, resulting in a series of valuable mutant enzymes. These dehydrogenases and aldolases are then combined to form an enzyme catalytic system, which is applied to the biosynthesis of 2,4-dihydroxybutyric acid.

[0007] The dehydrogenases and mutants provided by this invention exhibit stronger affinity for the substrates lactate and 2-keto-4-hydroxybutyric acid (2-keto-4-hydroxybutyric acid), enabling rapid utilization of low concentrations of 2-keto-4-hydroxybutyric acid in the reaction system. These enzymes possess a broad substrate spectrum, capable of dehydrogenation and hydrogenation reactions using various α-hydroxy acids or α-keto acids such as lactate, pyruvate, 2-keto-4-hydroxybutyric acid, and 2,4-dihydroxybutyric acid as substrates. The dehydrogenase bioelement is as follows: D1, D2, or D3:

[0008] D1: Dehydrogenase; D2: Nucleic acid sequence encoding the dehydrogenase; D3: Expression element containing the nucleic acid sequence, recombinant vector, or recombinant microorganism.

[0009] The protein sequence of the dehydrogenase is any one of the following E1-E6 or a mutant protein based on E3:

[0010] E1: Protein represented by SEQ ID NO: 1

[0011] E2: Protein shown in SEQ ID NO: 2

[0012] E3: Protein shown in SEQ ID NO: 3

[0013] E4: Protein shown in SEQ ID NO: 4

[0014] E5: Proteins with the same function obtained by mutating one or more amino acids, or by insertion and deletion, any of the E1-E4 proteins.

[0015] More preferably, based on E3, a single-point mutation is performed, specifically corresponding to the mutation of leucine (L) at site 7 to isoleucine (I) in amino acids 1 to 135 of SEQ ID NO: 3, the mutation of aspartic acid (D) at site 20 to phenylalanine (F), the mutation of glutamine (Q) at site 28 to serine (S), the mutation of glutamic acid (E) at site 41 to tryptophan (W), the mutation of methionine (M) at site 68 to aspartic acid (D), the mutation of threonine (T) at site 110 to lysine (K), the mutation of serine (S) at site 114 to tyrosine (Y), the mutation of histidine (H) at site 116 to isoleucine (I), and the mutation of histidine (H) at site 118 to lysine (I). Methionine (M) mutates to isoleucine (I), glutamic acid (E) at position 132 mutates to methionine (M), tyrosine (Y) at position 135 mutates to phenylalanine (F), histidine (H) at position 139 mutates to proline (P), aspartic acid (D) at position 160 mutates to tryptophan (W), histidine (H) at position 161 mutates to isoleucine (I), threonine (T) at position 180 mutates to tyrosine (Y), glutamic acid (E) at position 199 mutates to isoleucine (I), and serine (S) at position 208 mutates to aspartic acid (D). The following mutations were observed: Aspartic acid (D) at position 217 was mutated to methionine (M); tyrosine (Y) at position 219 was mutated to tryptophan (W); serine (S) at position 220 was mutated to alanine (A); phenylalanine (F) at position 221 was mutated to tyrosine (Y); methionine (M) at position 224 was mutated to isoleucine (I); serine (S) at position 235 was mutated to alanine (A); aspartic acid (D) at position 240 was mutated to methionine (M) or arginine (R); glutamic acid (E) at position 243 was mutated to methionine (M); and at position 259... Histidine (H) at position 261 is mutated to leucine (L), asparagine (N) at position 261 is mutated to glycine (G), aspartic acid (D) at position 264 is mutated to glycine (G), histidine (H) at position 281 is mutated to valine (V), histidine (H) at position 282 is mutated to glutamic acid (E), aspartic acid (D) at position 302 is mutated to isoleucine (I), glutamine (Q) at position 305 is mutated to leucine (L), alanine (A) at position 307 is mutated to leucine (L), and glutamic acid (E) at position 312 is mutated to leucine (L).

[0016] E6: Add a fusion tag to the N-terminus or C-terminus of any protein from E1 to E5, such as Poly-His, c-myc, Poly-Arg, GST, Flag, MBP, or Strep.

[0017] The present invention also provides an application of using aldolase and the above-mentioned dehydrogenase for the biosynthesis of 2,4-dihydroxybutyric acid.

[0018] The aldolase described is an aldolase capable of catalyzing the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyrate; specifically, it is a type I or type II aldolase; a specific example is 4-hydroxy-2-oxovalerate aldolase (Uniprot entry A0A7D8V3B9). The dehydrogenase described is a dehydrogenase capable of using L-lactic acid and 2-keto-4-hydroxybutyrate as substrates, specifically any of the E1-E6 dehydrogenases mentioned above.

[0019] Furthermore, by overexpressing aldolase and dehydrogenase genes in the host, 2,4-dihydroxybutyric acid is synthesized via whole-cell catalysis or microbial fermentation after the addition of substrate.

[0020] Furthermore, the plasmids pRSFDuet-1, pCDFDuet-1, pETDuet-1, pACYCDuet-1, and pCOLADuet-1 co-express aldolase and dehydrogenase, or the aldolase and dehydrogenase are respectively in two compatible plasmids, such as one in pTrc99a and the other in pTrc33a, and are co-transformed into the host strain.

[0021] More preferably, expression plasmids for aldolase and dehydrogenase are constructed using the pET vector and transformed into BL21(DE3) series expression strains. Crude enzyme solution is obtained by overexpression or pure enzyme is obtained by purification.

[0022] Furthermore, aldolase and dehydrogenase were combined in vitro and reacted with the substrate in a one-pot reaction to synthesize 2,4-dihydroxybutyric acid.

[0023] Preferably, the reaction system is supplemented with the following component: 0.5-10 mM NAD + 0.1-5 mM MgCl2, 5-50 mM Tris, HEPES, MOPS, Tricine, phosphate buffer, ammonia-ammonium chloride buffer, etc., to maintain the pH at 5-10, can obtain better catalytic performance.

[0024] Preferably, formaldehyde is added in batches at a concentration of less than 50 mM, or added slowly in a fed-batch manner, which improves the synthesis of 2,4-dihydroxybutyric acid using an in vitro catalytic system.

[0025] Preferably, the dehydrogenase is selected at 2 mg / ml, the aldolase at 1 mg / ml, and the in vitro catalytic system is more effective in synthesizing 2,4-dihydroxybutyric acid.

[0026] Beneficial effects:

[0027] This invention, through the exploration of the lactate dehydrogenase family, modifies the key active residues of dehydrogenases that simultaneously possess mixed catalytic activities of L-lactic acid and 2-keto-4-hydroxybutyrate to obtain mutants with enhanced activity. This results in dehydrogenases with extremely high affinity for lactate, pyruvate, and 2-keto-4-hydroxybutyrate, exhibiting affinity increases of tens of times compared to the highest-activity dehydrogenase reported in the literature, with substrate affinity at the micromolar level.

[0028] This invention introduces a novel dehydrogenase obtained through mining to construct a highly efficient 2,4-dihydroxybutyric acid (DHB) biosynthesis system, achieving a maximum DHB yield of 958 mM (115 g / L) and a conversion rate of >95% for both substrates, which is more than 100 times higher than the original technology. Attached Figure Description

[0029] The invention is further described in the following non-limiting drawings:

[0030] Figure 1 Cluster analysis diagram of dehydrogenases (SSNs).

[0031] Figure 2 SDS-PAGE images of dehydrogenase expression and purification: Left 1: Ldh4 precipitate, Left 5: Ldh5 precipitate, Right 4: Ldh1 purified protein, Right 3: Ldh2 purified protein; Right 4: Ldh3 purified protein, Right 5: Ldh6 purified protein.

[0032] Figure 3 Flowchart of the biosynthesis of 2,4-dihydroxybutyric acid involving aldolase and dehydrogenase. 1 represents L-lactic acid, 2 represents formaldehyde, 3 represents pyruvate, 4 represents 2-keto-4-hydroxybutyric acid, 5 represents 2,4-dihydroxybutyric acid, Ldh represents dehydrogenase, and Ald represents aldolase.

[0033] Figure 4 Relative activity data for the synthesis of 2,4-dihydroxybutyric acid by combinations of aldolase and different dehydrogenases.

[0034] Figure 5 HPLC chromatograms of the products and starting materials analyzed within 24 hours of the synthesis of 2,4-dihydroxybutyric acid using an enzyme cascade system (formaldehyde added in batches).

[0035] Figure 6 Graph showing the process of synthesizing 2,4-dihydroxybutyric acid using an enzyme cascade system (formaldehyde added in batches). Example

[0036] The invention will now be described in the following non-limiting embodiments.

[0037] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0038] Unless otherwise specified, all experimental or measurement methods used in this invention shall be performed as follows:

[0039] (1) Determination of 2,4-dihydroxybutyric acid and L-lactic acid: After the enzyme reaction, the protein was denatured by heating at 95 degrees Celsius, the denatured enzyme was removed by centrifugation, the sample was filtered through a 0.22 μm filter membrane and diluted to a concentration below 50 mM. HPLC analysis was performed using an Aminex HPX-87H chromatograph (300*7.8 mm). The mobile phase was 5 mM H2SO4, the flow rate was 0.6 mL / min, the column temperature was 15 °C, and the detection was performed using a RID differential detector at 35 °C.

[0040] (2) The culture medium used in this invention is LB medium (1L): 10g peptone, 10g sodium chloride, 5g yeast extract, pH 7.0.

[0041] (3) Protein overexpression culture conditions:

[0042] Seed culture: Inoculate single clones into 4 mL of LB medium, add ampicillin (100 μg / mL) according to the resistance of the transformed plasmid, and culture overnight at 37℃ and 220 rpm.

[0043] Expanded culture and expression: Transfer 4 mL of bacterial culture to 800 mL of resistant LB medium and incubate at 37°C and 220 rpm for 3-4 hours until OD500. 600 Adjust the concentration to 0.6-0.8. Cool the shaker to 16°C and allow the bacterial culture to cool (approximately 1 hour). Add a final concentration of 0.2 mM IPTG and incubate overnight at 16°C and 230 rpm for 16-20 hours. Collect the bacterial cells at 6000 rpm for 15 minutes.

[0044] Example 1: Dehydrogenase gene mining

[0045] Cluster analysis and gene selection methods for dehydrogenases

[0046] Cluster analysis of the L-lactate dehydrogenase superfamily (EC: 1.1.1.27) was performed using the EFI-EST tool (https: / / efi.igb.illinois.edu / efi-est / ), a protein sequence similarity network technology. Figure 1To facilitate expression in the E. coli system, we retained only bacterial, archaea, and fungal species, and divided them into different clusters by setting reasonable thresholds. We selected one gene from each cluster, for a total of 6 genes, for whole-genome synthesis. Specific gene information is shown in Table 1.

[0047] Table 1 shows the selected dehydrogenase sequences.

[0048] Serial Number enzyme name source Uniprot ID 1 Ldh1 Aureobasidium melanogenum A0A074VPE4 2 Ldh2 Listeria ivanovii subsp.londoniensis A0A097B5Y0 3 Ldh3 Staphylococcus intermedius A0A380G5Z1 4 Ldh4 Halarcobacteranaerophilus A0A4Q0Y181 5 Ldh5 Methanothermobacter sp.EMTCatA1 A0A223YW78 6 Ldh6 Mycoplasmopsissynoviae Q4A5V1

[0049] Example 2: Plasmid construction, expression, purification, and in vitro enzyme activity characterization of dehydrogenases

[0050] (1) Plasmid construction and preparation of expression strains

[0051] A series of target genes from the sequence listing in Example 1 were constructed into the pET16b vector through whole-gene synthesis and transformed into the BL21(DE3) expression strain to obtain a recombinant strain.

[0052] (2) Bacterial culture and induced expression

[0053] Inoculate the single clone into 4 mL of LB medium, add ampicillin (100 μg / mL) according to the resistance of the transformed plasmid, and incubate overnight at 37°C and 220 rpm.

[0054] Expanded culture and expression: Transfer 4 mL of bacterial culture to 800 mL of resistant LB medium and incubate at 37°C and 220 rpm for 3-4 hours until OD500. 600 Adjust the concentration to 0.6-0.8. Cool the shaker to 16°C and allow the bacterial culture to cool (approximately 1 hour). Add a final concentration of 0.2 mM IPTG and incubate overnight at 16°C and 230 rpm for 16-20 hours. Collect the bacterial cells at 6000 rpm for 15 minutes.

[0055] (3) Protein purification

[0056] Protein purification was performed using His-tag chelating resin and magnetic beads. The obtained proteins were then desalted to remove excess imidazole and finally stored in a storage solution of 20 mM HEPES, 300 mM NaCl, 10% glycerol, pH 7.5. Activity testing was subsequently performed, or the proteins were cryopreserved at -80°C. Four protein solutions were obtained through protein expression purification: Ldh1, Ldh2, Ldh3, and Ldh6. However, Ldh4 and Ldh5 primarily formed inclusion bodies. SDS-PAGE results are shown below. Figure 2 .

[0057] (4) Enzyme activity assay

[0058] The enzymatic activity of the dehydrogenase was determined by monitoring the initial consumption of NADH at 340 nm using a SpectraMax 190 microplate reader to measure the rate of the enzymatic reaction. The reaction rate was determined using the "Dynamic NADH Assay and PathCheck" method provided by SoftMax Pro7 software. 6.22 mM NADH was used. -1 cm -1 The NADH extinction coefficient was used to convert the results from OD / min to μM / min. At room temperature, in a 96-well UV-Vis transparent microplate, the reaction was initiated with NADH (0.5 mM) and, according to experimental requirements, with the addition of an appropriate concentration of enzyme and 10 mM substrate (pyruvate, 2-keto-4-hydroxybutyrate). The reaction rate was determined by real-time monitoring of the 314 nm absorbance (NADH concentration).

[0059] The activity data of the four purified soluble proteins are shown in Table 2. Under the condition of 10 mM substrate, Ldh6 has the highest enzyme activity for both substrates.

[0060] Table 2. Activity data of dehydrogenases catalyzing pyruvate and 2-keto-4-hydroxybutyrate.

[0061] Serial Number enzyme name <![CDATA[k for pyruvic acid cat (s -1 )]]> <![CDATA[k of 2-keto-4-hydroxybutyric acid cat (s -1 )]]> 1 Ldh1 0.038±0.002 0.064±0.004 2 Ldh2 0.14±0.01 0.15±0.01 3 Ldh3 0.26±0.02 0.53±0.02 6 Ldh6 21±1.0 28±3.0

[0062] Example 3: In vitro enzyme-catalyzed synthesis of 2,4-dihydroxybutyric acid by aldolase and different dehydrogenases

[0063] The selected aldolase was one capable of catalyzing the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyrate; a specific example was 4-hydroxy-2-oxovalerate aldolase (Uniprot entry A0A7D8V3B9), named 2-Ald11. For the dehydrogenase, the mutant Ldh0 (Uniprot ID: P04034, Q85C), a lactate dehydrogenase modified with 2-keto-4-hydroxybutyrate as a substrate, was used as a control. Following the same plasmid construction, protein expression, and purification steps as in Example 2, the two soluble proteins mentioned above were obtained. The differences in the ability of the different dehydrogenases Ldh1, Ldh2, Ldh3, Ldh6, and Ldh0 combined with aldolase 2Ald11 to catalyze the synthesis of 2,4-dihydroxybutyrate in an in vitro multi-enzyme system were tested. Figure 3 This is a flowchart illustrating the biosynthesis of 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid) involving aldolase and dehydrogenase. The reaction conditions for the enzyme cascade system synthesis of 2,4-dihydroxybutyric acid (with formaldehyde added in batches) are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-lactic acid, 2 mM NAD. +5mM MgCl2, 1mg / ml 2-Ald11, and 2mg / ml different Ldh were added, with 50mM formaldehyde added every 30 minutes for a total of 10 additions. The amount of 2,4-dihydroxybutyric acid generated was monitored by HPLC analysis. The combination of 2-Ald11 and Ldh0 was used as a control, with its activity set to 1. The other combinations were compared with this control as relative activities. The specific relative activity data for different combinations are shown in [link to relevant data]. Figure 4 It can be seen that Ldh3 increased the production of 2,4-dihydroxybutyric acid by 1.7 times compared with the control dehydrogenase Ldh0.

[0064] Example 4: Enzyme kinetic characterization of Ldh3 dehydrogenase

[0065] Observed k of Ldh3 cat While its affinity is not high, it performs best in in vitro multi-enzyme catalysis systems. Therefore, its affinity for the substrates L-lactic acid, pyruvate, and 2-keto-4-hydroxybutyrate was investigated. Enzyme kinetics of the dehydrogenase were characterized by measuring the initial consumption of NADH at 340 nm using a SpectraMax 190 microplate reader to determine the enzymatic reaction rate. The reaction rate was determined using the "Dynamic NADH Assay and PathCheck" method provided by SoftMax Pro7 software. The results were converted from OD / min to μM / min using the NADH extinction coefficient of 6.22 mM⁻¹ cm⁻¹. At room temperature, in 96-well UV-Vis transparent microplates, the reaction was initiated with NADH (0.5 mM) and, according to experimental requirements, appropriate concentrations of enzyme and different concentrations of substrates (pyruvate, 2-keto-4-hydroxybutyrate). The reaction rate was determined by real-time monitoring of the 314 nm absorbance (NADH concentration). For L-lactic acid, add 2 mM NAD. + Instead of the 0.5 mM NADH mentioned above, the enzyme reaction was carried out. The k of the aldolase was determined by fitting the data to the standard Michaelis-Menten equation, the Hill equation, or an equation with substrate inhibition. cat K m and K i Values. The enzyme kinetic properties for different substrates are shown in Table 3. Kinetic data show that Ldh3 exhibits extremely strong affinity for a variety of substrates, especially for 2-keto-4-hydroxybutyric acid (K+). m It is 52 μM, compared to the literature ( CJR;Topham,CM;Malbert,Y.; JM; Walther, T., Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway. Biochemical Journal 2018, 475(23), 3887-3901.) reported the optimal dehydrogenase mutant (K) optimized using 2-keto-4-hydroxybutyric acid as a substrate. m The affinity for substrates (1.6 mM) was increased by 30 times. Strong substrate affinity is a key factor for the optimal activity of this enzyme in multi-enzyme systems and is also an important characteristic of this enzyme. Strong substrate affinity can effectively utilize low concentrations of intermediates in the system, enabling the entire multi-enzyme catalytic system to operate efficiently.

[0066] Table 3: Enzyme kinetic data of dehydrogenase Ldh3 on different substrates

[0067] Substrate <![CDATA[k cat (s -1 )]]> <![CDATA[K m (mM)]]> <![CDATA[K i (mM)]]> L-lactic acid <![CDATA[(4.3±0.1)×10 -3 ]]> 0.66±0.07 No inhibition Pyruvic acid 1.0±0.1 0.029±0.007 2.5±0.6 2-Keto-4-hydroxybutyric acid 0.71±0.01 0.052±0.004 30±3

[0068] Example 5: Design, construction and activity assay of dehydrogenase mutants

[0069] To improve the thermostability of dehydrogenases and ensure their storage capacity and reusability, we used the Fireprot online tool, specifically developed for stable proteins, to design mutants of Ldh3. Two main principles were followed: predicting mutants from an evolutionary perspective based on conservation, and predicting mutants from a binding energy perspective based on Rosetta calculations. A total of 35 single-point mutants were designed. These single-point mutants of Ldh3 dehydrogenase were constructed using overlap PCR. Following the same plasmid construction, protein expression, purification, and activity assay procedures as in Example 2, soluble proteins of the above mutants were obtained, and their activities were measured. Specific mutation sites and types, as well as activity data, are shown in Table 4. The E243M mutant showed improved activity to different substrates, while the Y219W, D217M, and M224I showed a reversal of their substrate preferences. Furthermore, the thermostability of the mutants was significantly altered; the wild-type enzyme became turbid at 50℃, while the mutants showed no significant changes at 50℃ or even 55℃.

[0070] Table 4: Ldh3 dehydrogenase mutants and relative activity data

[0071]

[0072]

[0073] Example 6: Synthesis of 2,4-dihydroxybutyric acid using an enzyme cascade system (formaldehyde added in batches)

[0074] The in vitro multi-enzyme catalytic system for the synthesis of 2,4-dihydroxybutyric acid was tested under multiple conditions. Formaldehyde was added in batches. Specific enzyme reaction conditions were as follows: 5-50 mM pH 6.5, 8.5, 9.0 buffer, 0.2 M L-lactic acid, 0.5-2 mM NAD. + 5 mg / ml MgCl₂, 1 mg / ml 2-Ald¹⁺, and 2 mg / ml Ldh₃ were reacted at 40℃. 50 mM formaldehyde and 50 mM L-lactic acid were added every 30 min until both formaldehyde and L-lactic acid reached a final concentration of 1 M. The amount of DHB generated and the remaining feedstock at different time points were monitored by HPLC analysis. The highest DHB yield reached 958 mM after 24 h. The HPLC chromatogram of the highest yield group is shown below. Figure 5 The process curves for real-time monitoring of substrate and product changes are shown in the figure. Figure 6 In the first 8 hours, the yield of 2,4-DHB reached as high as 14.2 g / L / h, but the enzyme reaction rate subsequently decreased. After 10 hours, the total sample volume reached 1 M, and substrate replenishment was stopped. At this point, the concentration of 2,4-DHB reached 828 mM. However, the cumulative formaldehyde concentration in the system reached 192 mM, causing partial protein denaturation. After being left overnight to 24 hours, the highest yield of 2,4-DHB reached 958 mM, and the conversion rates of both substrates were >95%.

[0075] Example 7: Synthesis of 2,4-dihydroxybutyric acid by microbial fermentation or whole-cell catalysis

[0076] Construction of functional plasmids and bacterial strains:

[0077] The pathway-related plasmids pRSFDuet-1-2-Ald11-Ldh3, pCDFDuet-1-2-Ald11-Ldh3, pETDuet-1-2-Ald11-Ldh3, pACYCDuet-1-2-Ald11-Ldh3, pCOLADuet-1-2-Ald11-Ldh3, pTrc99a-2-Ald11, pTrc33a-Ldh3, pTrc33a-2-Ald11, and pTrc99a-Ldh3 were constructed. Homologous primers were designed and assembled using the Gibson method.

[0078] Construction of strains: The key gene frmA in the formaldehyde detoxification system of strains BL21(DE3) and W3110 was knocked out to form functional strains BL21(DE3)△frmA and W3110△frmA.

[0079] Construction of recombinant strains: The five vectors pRSFDuet-1-2-Ald11-Ldh3, pCDFDuet-1-2-Ald11-Ldh3, pETDuet-1-2-Ald11-Ldh3, pACYCDuet-1-2-Ald11-Ldh3, and pCOLADuet-1-2-Ald11-Ldh3 were transformed into either BL21(DE3) or BL21(DE3)△frmA strains to form recombinant strains. The vectors pTrc99a-2-Ald11 and pTrc33a-Ldh3, or pTrc33a-2-Ald11 and pTrc99a-Ldh3, were transformed into W3110△frmA strains to form recombinant strains.

[0080] Furthermore, the recombinant strain was seed cultured to obtain whole cells through an induced expression process, followed by enzymatic reactions. The whole-cell reaction system was as follows: 50 mM pH 6.5 phosphate buffer, 0.2 M L-lactic acid, 0.5 mM NAD+. + 5 mM MgCl2, 10 mg / ml wet cells, and 50 mM formaldehyde were added every 1 hour for a total of 4 times. The amount of 2,4-dihydroxybutyric acid synthesized was tested.

[0081] The results showed that 7-50 mM of 2,4-dihydroxybutyric acid could be produced in different recombinant strain systems.

[0082] Another approach involves culturing the recombinant strain directly at 37 degrees Celsius, adding 0.2 mM IPTG after the OD600 reaches 0.4, then cooling to 30 degrees Celsius and culturing for 3 hours. After that, the substrates formaldehyde and L-lactic acid are added at a rate of 50 mM / h each time, for a total of 10 additions, and the amount of 2,4-dihydroxybutyric acid synthesized is then tested.

[0083] The results showed that 15-60 mM of 2,4-dihydroxybutyric acid could be produced in different recombinant strain systems.

Claims

1. Application of novel dehydrogenases and their mutants as enzyme elements. These enzymes have a broad substrate spectrum, capable of using various α-keto acids or α-hydroxy acids such as L-lactic acid, pyruvate, 2-keto-4-hydroxybutyric acid, and 2,4-dihydroxybutyric acid as substrates. Specifically, two reactions are applied: first, a dehydrogenation reaction using L-lactic acid as a substrate; second, a hydrogenation reaction using 2-keto-4-hydroxybutyric acid as a substrate, which is then combined with aldolase to synthesize 2,4-dihydroxybutyric acid. The dehydrogenase biological elements are D1, D2, or D3 as follows: D1: Dehydrogenase; D2: Nucleic acid sequence encoding the dehydrogenase; D3: Expression element containing the nucleic acid sequence, recombinant vector, or recombinant microorganism. The protein sequence of the dehydrogenase is any one of the following E1-E6 or a mutant protein based on E3: E1: Protein shown in SEQ ID NO: 1 E2: Protein shown in SEQ ID NO: 2 E3: Protein shown in SEQ ID NO: 3 E4: Protein shown in SEQ ID NO: 4 E5: Proteins with the same function obtained by mutating one or more amino acids, or by insertion and deletion, any protein from E1 to E4. More preferably, based on E3, a single-point mutation is performed, specifically corresponding to the mutation of leucine (L) at site 7 to isoleucine (I) in amino acids 1 to 135 of SEQ ID NO: 3, the mutation of aspartic acid (D) at site 20 to phenylalanine (F), the mutation of glutamine (Q) at site 28 to serine (S), the mutation of glutamic acid (E) at site 41 to tryptophan (W), the mutation of methionine (M) at site 68 to aspartic acid (D), the mutation of threonine (T) at site 110 to lysine (K), the mutation of serine (S) at site 114 to tyrosine (Y), and the mutation of threonine (T) at site 116 to lysine (K). Histidine (H) mutated to isoleucine (I), methionine (M) at position 118 mutated to isoleucine (I), glutamic acid (E) at position 132 mutated to methionine (M), tyrosine (Y) at position 135 mutated to phenylalanine (F), histidine (H) at position 139 mutated to proline (P), aspartic acid (D) at position 160 mutated to tryptophan (W), histidine (H) at position 161 mutated to isoleucine (I), threonine (T) at position 180 mutated to tyrosine (Y), glutamic acid (E) at position 199 mutated to isoleucine (I), and at position 208... Serine (S) mutates to aspartic acid (D), aspartic acid (D) at position 217 mutates to methionine (M), tyrosine (Y) at position 219 mutates to tryptophan (W), serine (S) at position 220 mutates to alanine (A), phenylalanine (F) at position 221 mutates to tyrosine (Y), methionine (M) at position 224 mutates to isoleucine (I), serine (S) at position 235 mutates to alanine (A), aspartic acid (D) at position 240 mutates to methionine (M) or arginine (R), and glutamic acid (E) at position 243 mutates to methionine. The following mutations were made: histidine (H) at position 259 was mutated to leucine (L); asparagine (N) at position 261 was mutated to glycine (G); aspartic acid (D) at position 264 was mutated to glycine (G); histidine (H) at position 281 was mutated to valine (V); histidine (H) at position 282 was mutated to glutamic acid (E); aspartic acid (D) at position 302 was mutated to isoleucine (I); glutamine (Q) at position 305 was mutated to leucine (L); alanine (A) at position 307 was mutated to leucine (L); and glutamic acid (E) at position 312 was mutated to leucine (L). E6: Add a fusion tag to the N-terminus or C-terminus of any protein from E1 to E5. The tag includes, but is not limited to, Poly-His, c-myc, Poly-Arg, GST, Flag, MBP, and Strep.

2. The application according to claim 1, characterized in that: This can be carried out through recombinant microbial fermentation, whole-cell catalysis, or in vitro enzyme catalysis.

3. The application according to claim 2, characterized in that: The recombinant bacteria obtained by introducing the recombinant vector into the host bacteria are overexpressed to produce the dehydrogenase mentioned above, which participates in the dehydrogenation of lactate to produce pyruvate or the hydrogenation of 2-keto-4-hydroxybutyrate to produce 2,4-dihydroxybutyrate, as well as the reversible reaction process corresponding to the above reaction. The recombinant vector is a vector obtained by inserting the coding nucleic acid sequence of the dehydrogenase mentioned in claim 2 into the expression vector.

4. The application according to claim 1, characterized in that: The in vitro enzyme reaction system comprises any of the dehydrogenases in claim 1, wherein the dehydrogenase participates in the dehydrogenation of lactate to pyruvate or the hydrogenation of 2-keto-4-hydroxybutyrate to 2,4-dihydroxybutyrate, as well as the reversible reaction process corresponding to the above reaction.

5. A method for preparing 2,4-dihydroxybutyric acid, characterized in that, Including the following steps: (1) L-lactic acid is dehydrogenated to pyruvate by any of the dehydrogenases in claim 1; (2) Pyruvic acid and formaldehyde condense under the action of aldolase to form 2-keto-4-hydroxybutyric acid; (3) 2-keto-4-hydroxybutyric acid is dehydrogenated by any of the dehydrogenases in claim 1 to generate 2,4-dihydroxybutyric acid.

6. The method according to claim 5, characterized in that: This can be carried out through recombinant microbial fermentation, whole-cell catalysis, or in vitro enzyme catalysis.

7. The method according to claim 5, characterized in that: The recombinant bacteria obtained by introducing the recombinant vector into the host bacteria are overexpressed to produce the aldolase and dehydrogenase described above, and 2,4-dihydroxybutyric acid is synthesized through the three-step reaction described in claim 5; the recombinant vector contains at least one aldolase and at least one dehydrogenase as described in claim 1, and the nucleic acid sequences encoded by them are inserted into a vector obtained by inserting them into an expression vector or a combination of multiple coexisting expression vectors.

8. The method according to claim 5, characterized in that: An in vitro enzyme catalysis system, characterized in that: (1) At least one aldolase and at least one dehydrogenase protein of claim 1 are contacted with the reaction substrates lactic acid and formaldehyde to carry out a catalytic reaction, thereby obtaining the 2,4-dihydroxybutyric acid; (2) The in vitro multi-enzyme catalytic system contains formaldehyde, lactic acid, aldolase, and dehydrogenase; (3) Preferably, the reaction system is supplemented with the following component: 0.1-10mM NAD + 0.1-5mM MgCl2, 5-50mM Tris, HEPES, MOPS, Tricine, phosphate buffer, ammonia-ammonium chloride buffer, etc., to maintain the pH at 5-10, can obtain better catalytic performance; (4) More preferably, the temperature of the catalytic reaction is 20-60°C, more preferably, 25-45°C, and even more preferably, 35-40°C.

9. The method according to claim 8 improves the reusability of enzymes by introducing enzyme immobilization technology, wherein the immobilization technology is characterized by including but not limited to the covalent or non-covalent binding of tagged magnetic beads or resin or porous nanomaterials to proteins, metal-organic frameworks (MOFs) or HP-MOFs, metal inorganic frameworks, and self-assembled proteins such as protein nanocages.

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