Biosynthesis method of 2, 4-dihydroxybutyric acid

Abstract

The invention belongs to the technical field of biology, and discloses a biosynthesis method of 2, 4-dihydroxybutyric acid. According to the biosynthesis method, L-malic acid and formaldehyde serve as substrates to synthesize 2, 4-dihydroxybutyric acid, L-malic acid is converted into oxaloacetic acid through L-malic dehydrogenase, oxaloacetic acid is decarboxylated to generate pyruvic acid, pyruvic acid and formaldehyde are condensed under the action of aldolase to generate 2-ketone-4-hydroxybutyric acid, and 2, 4-dihydroxybutyric acid is converted into 2, 4-dihydroxybutyric acid through dehydrogenase. The invention relates to 2, 4-dihydroxybutyric acid. The method can be realized through an in-vitro multi-enzyme catalysis system, and can also be completed by introducing a way into microorganisms. Furthermore, the formaldehyde can be generated from methanol, formic acid or sarcosine under the action of enzyme. Therefore, the monocarbon resources such as methanol and formaldehyde can be introduced to synthesize the 2, 4-dihydroxybutyric acid with high value, the raw materials are low in price, and the whole synthesis method has the advantages of high atom utilization rate and high conversion rate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for the biosynthesis of 2,4-dihydroxybutyric acid. Background Technology

[0002] 2,4-Dihydroxybutyric acid (DHB) can be synthesized in a one-step chemical conversion with methanethiol into the promising methionine analog 2-hydroxy-4-methylthiobutyric acid (HMTB). It can also serve as a precursor for the synthesis of other 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 as more applications are discovered. For example, γ-butyrolactone has been found to be used in paint stripping, circuit board cleaning, soy flavoring, and even as an organic solvent. Recently, metabolic pathways for synthesizing 1,3-propanediol, 1,2,4-butanetriol, and 1,4-butanediol from DHB have been developed. These compounds are bulk chemicals with wide applications as precursors for plastics, solvents, and aerospace propellants. Therefore, DHB is a multifunctional platform molecule with high industrial potential. However, its high price and limited availability by manufacturers significantly restrict its applications. Therefore, developing a low-cost and efficient method for DHB biosynthesis has significant industrial application value.

[0003] However, no natural biosynthetic pathways were found in DHB. Currently, the main feasible non-natural biosynthetic pathways are as follows: 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 a two-step enzymatic synthesis method using homoserine as a raw material, which involves the formation of 2-keto-4-hydroxybutyric acid by transaminase and its reduction to DHB by dehydrogenase. This method, using *E. coli* fermentation for 48 hours, yielded 5.3 g / L of DHB. 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-Sufficient Route. ACS Sustainable Chemistry & Engineering 2022, 10(43), 14361-14369.) increased the yield to 17.8 g / L (44 h) by introducing a cofactor recycling system. Li et al. (Li, X.; Cai, Z.; Li, Y.; Zhang, Y., Design and Construction of a Non-Natural Malate to 1,2,4-Butanetriol Pathway Creates Possibility to Produce 1,2,4-Butanetriol from Glucose. Scientific Reports 2014, 4(1), 5541.) used malic acid as a starting material, and activated the terminal carboxyl group of malic acid with coenzyme A, reducing it to an aldehyde group, and then reducing it to a hydroxyl group to form 2,4-DHB. However, this pathway only produced trace amounts of DHB (6.4 mg / L, 24 h). Similarly, Walther et al. (Walther, T.; Topham, CM; Irague, R.; Auriol, C.; Baylac, A.; Cordier, H.; Dressaire, C.; Lozano-Hug uet,L.;Tarrat,N.;Martineau,N.;Stodel,M.;Malbert,Y.;Maestracci,M.;Huet,R.;André,I.;Remaud-Siméon,M.; JM, Construction of a synthetic metabolic pathway for biosynthesis of the non-natural methionine precursor 2,4-dihydroxybutyricacid. Nature Communications 2017, 8(1), 15828.) developed a method for producing DHB using L-malic acid as a raw material, utilizing the hybrid activity of enzymes and enzyme modification, and employing malate kinase, malate semialdehyde dehydrogenase, and malate semialdehyde reductase in a three-step enzymatic reaction. The yield in engineered E. coli strains was 1.8 g / L over 24 hours. In 2023, Walther's team ( CJR; Wagner, N.; Rabe, K.; Walther, T., "Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol." Nature Communications 2023, 14(1), 1931.) reported a biosynthetic process of DHB from inexpensive C2 compound ethylene glycol, involving a six-step enzymatic reaction of ethylene glycol dehydrogenase, D-threonine aldolase, D-threonine dehydrogenase, D-threonine-1,4-lactonease, D-threonine dehydrogenase, and 2-oxo-4-hydroxybutyrate reductase. Based on this pathway, an engineered *E. coli* strain achieved a maximum yield of 0.8 g / L and 1 g / L, respectively, using ethylene glycol or glycolaldehyde as substrates over 24 hours. In addition, Li et al. (CN112921021A) designed a DHB biosynthetic route based on one-carbon raw materials formaldehyde or methanol, but the yield was less than 10 mM, approximately 1.2 g / L. Recently, Dong et al. (Dong, X.; Sun, C.; Guo, J.; Ma, X.; Xian, M.; Zhang, R., Highly efficient biosynthesis of 2,4-dihydroxybutyric acid by a methanol assimilation pathway in engineered Escherichia coli. Green Chemistry 2023, 25(19), 7662-7672.) developed a pathway for the production of DHB from glucose and C1 methanol via a three-step enzymatic reaction involving methanol dehydrogenase, aldolase, and dehydrogenase. The highest yield achieved in a 5L fermenter, using glucose and methanol as common substrates, was 14.6 g / L over 79 h. While the above route utilizes readily available and inexpensive raw materials, the conversion rate of the catalytic system is generally low, resulting in high costs. Therefore, developing a more economical and efficient biosynthesis method for 2,4-dihydroxybutyric acid has significant economic value. Summary of the Invention

[0004] To address the aforementioned issues, this invention presents a biosynthetic route for 2,4-dihydroxybutyric acid, using inexpensive L-malic acid as the initial raw material. Under the action of aldolase and dehydrogenase, L-malic acid undergoes condensation with formaldehyde, ultimately generating 2,4-dihydroxybutyric acid through several conversion steps.

[0005] This invention uses L-malic acid as a starting material and converts L-malic acid and formaldehyde into 2,4-dihydroxybutyric acid under the action of aldolase and dehydrogenase. The entire process releases only one molecule of carbon dioxide and produces no other toxic byproducts, which has high atom economy and is environmentally friendly.

[0006] The core approach of this invention is approach one, which uses L-malic acid and formaldehyde as raw materials to synthesize 2,4-dihydroxybutyric acid. The main steps are as follows: Figure 1 Pathway 1. (1) L-malate is dehydrogenated to oxaloacetic acid under the action of dehydrogenase; (2) Oxaloacetic acid is decarboxylated to pyruvate spontaneously or under the action of divalent cations or enzymes (oxaloacetic acid decarboxylase, aldolase, phosphoenolpyruvate kinase, malic acid kinase and pyruvate kinase); (3) Aldolase catalyzes the reaction of formaldehyde and pyruvate to generate 2-keto-4-hydroxybutyric acid; (4) Dehydrogenase catalyzes the hydrogenation of 2-keto-4-hydroxybutyric acid to generate 2,4-dihydroxybutyric acid.

[0007] Furthermore, the present invention also includes a method for preparing the substrate formaldehyde. Based on pathway one, it may further include methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase, etc., to form a cascade of enzymatic catalytic reactions that generate formaldehyde from other precursor molecules. That is, it may further include the following steps: methanol dehydrogenase catalyzes the formation of formaldehyde from methanol (see pathway two); formaldehyde dehydrogenase catalyzes the formation of formaldehyde from formic acid (see pathway three); sarcosine oxidase catalyzes the formation of formaldehyde from sarcosine (see pathway four).

[0008] A novel biosynthetic pathway for 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid) was constructed by combining the above-mentioned new approaches, utilizing compounds such as methanol, sarcosine, and formic acid. This pathway introduces abundant one-carbon compounds into the biosynthesis of 2,4-dihydroxybutyric acid, achieving advantages such as reduced production costs, increased molar conversion rate, and atom utilization. The 2,4-dihydroxybutyric acid multi-enzyme reaction system and microbial cell factory established based on this novel pathway have significant application prospects.

[0009] Therefore, the present invention provides a method for the biosynthesis of 2,4-dihydroxybutyric acid, wherein the catalytic system of the method comprises an aldolase and a dehydrogenase; further derived pathways include methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase. More preferably, the addition of oxaloacetate decarboxylase improves the efficiency of the catalytic system.

[0010] This invention provides a biosynthetic method for synthesizing 2,4-dihydroxybutyric acid (2,4-DHA) using L-malic acid and methanol / formaldehyde / formic acid / sarcosine as raw materials. The method involves catalytic synthesis of 2,4-DHA through an in vitro multi-enzyme system containing various enzymes required for the reaction, or through whole-cell or microbial fermentation using recombinant strains expressing these enzymes.

[0011] Furthermore, the aldolase and dehydrogenase genes are overexpressed in the host. Preferably, the oxaloacetate decarboxylase gene is introduced. Further derivative pathways also include methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase genes. After the addition of substrate, 2,4-dihydroxybutyric acid is synthesized through whole-cell catalysis or microbial fermentation.

[0012] More preferably, expression plasmids of the above genes are constructed using the pET vector and transformed into BL21(DE3) series expression strains, and crude enzyme solution is obtained by overexpression or pure enzyme is obtained by purification.

[0013] Furthermore, aldolase and dehydrogenase are combined in vitro and reacted with the substrate in a one-pot reaction to synthesize 2,4-dihydroxybutyric acid. Preferably, oxaloacetate decarboxylase is introduced, and further derivative pathways include methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase.

[0014] Preferably, the reaction system in pathway one contains the following components: 5-50 mM buffer solution, pH between 6 and 10, and 0-10 mM NAD. + 0.5-10mM MgCl2 or other divalent metal ions, 0.5-2mg / ml aldolase and 1-3mg / ml dehydrogenase, and 50-500mM formaldehyde are used to catalyze the reaction to obtain 2,4-dihydroxybutyric acid.

[0015] Preferably, formaldehyde is added in batches at a concentration of 10-50 mM, or added slowly in a continuous flow manner, which improves the synthesis of 2,4-dihydroxybutyric acid using an in vitro catalytic system.

[0016] Preferably, the reaction system in pathway two contains the following components: 5-50 mM buffer solution, pH 6-10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + It is used to catalyze the reaction with 0.5-2 mg / ml aldolase, 1-3 mg / ml dehydrogenase, and 1-4 mg / ml methanol dehydrogenase, 10-500 mM methanol, to obtain 2,4-dihydroxybutyric acid.

[0017] Preferably, the reaction system in pathway three contains the following components: 5-50 mM buffer solution, pH 6-10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + It also contains 0.5-2 mg / ml aldolase, 1-3 mg / ml dehydrogenase, 1-4 mg / ml formaldehyde dehydrogenase, and 5-500 mM formic acid, which catalyze a reaction to obtain 2,4-dihydroxybutyric acid.

[0018] Preferably, the reaction system in pathway four contains the following components: 5-50 mM buffer solution, pH 6-10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + It also contains 0.5-2 mg / ml aldolase, 1-3 mg / ml dehydrogenase, 1-4 mg / ml sarcosine oxidase, and 5-500 mM sarcosine, which catalyze a reaction to obtain 2,4-dihydroxybutyric acid.

[0019] More preferably, adding 0.5-2 mg / ml oxaloacetate decarboxylase to the reaction system based on the conditions described in routes one, two, three, and four can increase the production rate and yield of 2,4-dihydroxybutyric acid.

[0020] This invention further provides a genetically engineered recombinant strain for producing 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid). The recombinant strain overexpresses dehydrogenases and aldolases in host cells, preferably overexpressing oxaloacetate decarboxylase, thereby synthesizing 2,4-dihydroxybutyric acid. Further, it may include overexpression of methanol dehydrogenase, formaldehyde dehydrogenase, or sarcosine oxidase in the recombinant strain, thus enabling the conversion of compounds such as methanol, formic acid, or sarcosine into formaldehyde. Simultaneously, it may also include overexpression of enzymes related to the L-malate pathway in the recombinant strain, i.e., introducing the novel pathway of this invention based on a high-yield L-malate engineered strain, with the high-yield strain providing the ability to synthesize L-malate.

[0021] In one embodiment of the present invention, the aldolase is an aldolase capable of catalyzing the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyric acid; a specific example is 4-hydroxy-2-oxovalerate aldolase (Uniprot entryA0A7D8V3B9, SEQ ID No.4).

[0022] In one embodiment of the present invention, the dehydrogenase is capable of using L-malic acid and 2-keto-4-hydroxybutyric acid as substrates, specifically L-malic acid dehydrogenase (EC: 1.1.1.37) and its mutants, more specifically a five-point mutant (SEQ ID No. 5) of Escherichia coli L-malic acid dehydrogenase (Uniprot entry P61889).

[0023] In one embodiment of the present invention, the methanol dehydrogenase is a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase, an NAD-dependent methanol dehydrogenase, or a methanol oxidase. A specific example selected is an NAD-dependent methanol dehydrogenase (Uniprot entry P42327, SEQ ID No. 6).

[0024] In one embodiment of the present invention, the sarcosine oxidase (EC: 1.5.3.1) is capable of catalyzing the production of formaldehyde and glycine from sarcosine, a specific example being (Uniprot entry P40859, SEQ ID No. 7).

[0025] In one embodiment of the present invention, the formaldehyde dehydrogenase (EC: 1.2.1.46) can catalyze the formation of formaldehyde from formic acid, specifically (Uniprot entry P46154, SEQ ID No. 8).

[0026] In one embodiment of the present invention, the oxaloacetate decarboxylase (EC:4.1.1.112) can catalyze the decarboxylation of oxaloacetate to produce pyruvate and carbon dioxide. A specific example is Pseudomonas aeruginosa oxaloacetate decarboxylase (Uniprotentry Q9HUU1, SEQ ID No.9).

[0027] This invention provides a novel biosynthetic method for 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid). The method involves a basic pathway comprising dehydrogenase and aldolase elements. A novel pathway for the synthesis of 2,4-dihydroxybutyric acid from formaldehyde and L-malic acid has been successfully constructed using either an in vitro multi-enzyme reaction system or by overexpressing two enzyme elements in recombinant microorganisms. Based on this basic pathway, it can further include enzyme elements catalyzing the synthesis of formaldehyde, such as methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase. A pathway for the conversion of methanol, sarcosine, or formic acid to 2,4-dihydroxybutyric acid has been successfully constructed using either an in vitro multi-enzyme reaction system or by overexpressing three basic enzyme elements—methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase—or dehydrogenase and aldolase in recombinant microorganisms. Furthermore, the introduction of an oxaloacetate decarboxylase element accelerates the decarboxylation process of oxaloacetate, making the pathway faster and more efficient.

[0028] In summary, this invention introduces high-energy-density, low-cost one-carbon compounds such as methanol, formaldehyde, and formic acid into the synthesis route of 2,4-dihydroxybutyric acid, resulting in low cost and significant application prospects. Attached Figure Description

[0029] Figure 1 , 2 A schematic diagram of a new synthetic route for 4-dihydroxybutyric acid.

[0030] Figure 2 HPLC chromatogram of the synthesis of 2,4-dihydroxybutyric acid using formaldehyde, methanol, formic acid, sarcosine, and L-malic acid as substrates via an in vitro multi-enzyme system. Detailed Implementation

[0031] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0032] Unless otherwise specified, the experimental or measurement methods used in this invention shall be performed as follows:

[0033] For the determination of 2,4-dihydroxybutyric acid and L-malic acid, after the enzyme reaction, the protein was denatured by heating at 95°C, the denatured enzyme was removed by centrifugation, the sample was filtered through a 0.22 μm filter membrane, and the concentration was diluted to 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.

[0034] The culture medium used in this invention is as follows:

[0035] (1) M9 Inorganic Salt Culture Medium: The basic components include (1L): 47.8mM Na2HPO4, 22mM M KH2PO4, 8.6mM NaCl, 93mM NH4Cl, 2mM MgSO4, 100μM CaCl2; Trace elements (1L): 134μM EDTA, 31μM FeCl3, 6.2μM ZnCl2, 0.76μM CuCl2, 0.42μM CoCl2, 1.62μM M H3BO3, 0.081μM MnCl2; Glucose: 20g / L, and other substances such as antibiotics and amino acids are added as needed.

[0036] (2) LB medium (1L): 10g peptone, 10g sodium chloride, 5g yeast extract, pH 7.0.

[0037] Protein overexpression culture conditions:

[0038] Seed culture: Inoculate a single clone into 4 mL of LB medium, add ampicillin (100 μg / mL) according to the resistance of the transformed plasmid, and culture overnight at 37°C and 220 rpm.

[0039] 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 reaches zero. 600Adjust 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. Perform enzymatic reactions using whole cells or purified protein.

[0040] Example 1: Screening of key enzymes and plasmid construction in the biosynthetic pathway of 2,4-dihydroxybutyric acid.

[0041] This invention introduces one-carbon compounds such as methanol / formaldehyde into the synthesis of 2,4-dihydroxybutyric acid. Through the dehydrogenation of L-malate by dehydrogenase, the decarboxylation of oxaloacetic acid, the aldol condensation of formaldehyde and pyruvate, and the hydrogenation of the intermediate 2-keto-4-hydroxybutyric acid by dehydrogenase, 2,4-dihydroxybutyric acid is finally synthesized. The basic synthetic route is shown below. Figure 1 Pathway 1: Using formaldehyde and L-malic acid as substrates, 2,4-dihydroxybutyric acid is synthesized by dehydrogenase and aldolase catalysis.

[0042] Aldolases are aldolases that can catalyze the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyric acid; a specific example is the aldolase of the genus *Saprophytica* (Uniprot entry A0A7D8V3B9, SEQ ID No. 4), named VhAld.

[0043] Dehydrogenases are enzymes that can use L-malic acid and 2-keto-4-hydroxybutyric acid as substrates. Specifically, L-malic acid dehydrogenase (EC: 1.1.1.37) and its mutants, more specifically, a five-point mutant (SEQ ID No. 5) of Escherichia coli L-malic acid dehydrogenase (Uniprot entry P61889) can better catalyze the hydrogenation of 2-keto-4-hydroxybutyric acid to 2,4-dihydroxybutyric acid. This mutant is named Mdh0.

[0044] Methanol dehydrogenase is a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase, an NAD-dependent methanol dehydrogenase, or a methanol oxidase. A specific example selected is the NAD-dependent methanol dehydrogenase from Bacillus stearothermophilus (Uniprotentry P42327, SEQ ID No. 6), named BsMdh.

[0045] Sarcosine oxidase (EC: 1.5.3.1) catalyzes the production of formaldehyde and glycine from sarcosine. A specific example is the sarcosine oxidase of Bacillus (Uniprot entry P40859, SEQ ID No. 7), named BsSoxA.

[0046] Formaldehyde dehydrogenase (EC: 1.2.1.46) can catalyze the formation of formaldehyde from formic acid. A specific example is the formaldehyde dehydrogenase of *Pseudomonas putida* (Uniprot entry P46154, SEQ ID No. 8), named PpfdhA.

[0047] Oxaloacetate decarboxylase (EC:4.1.1.112) can catalyze the decarboxylation of oxaloacetate to produce pyruvate and carbon dioxide. A specific example is Pseudomonas aeruginosa oxaloacetate decarboxylase (Uniprot entry Q9HUU1, SEQ ID No.9), named PaOadC.

[0048] (1) Plasmid construction and preparation of expression strains

[0049] The target genes in the sequence listings of SEQ ID No. 4-No. 9 in Example 1 were synthesized by Genewiz using whole-gene synthesis. Then, NdeI and BamHI were selected restriction enzyme sites to construct the pET16b vector, obtaining expression vectors pET16b-VhAld, pET16b-Mdh0, pET16b-BsMdh, pET16b-BsSoxA, pET16b-PpfdhA, and pET16b-PaOadC. The above vectors were transformed into the BL21(DE3) expression strain to obtain recombinant strains.

[0050] (2) Bacterial culture and induced expression

[0051] 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.

[0052] 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 reaches zero. 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.

[0053] (3) Protein purification

[0054] 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.

[0055] Example 2: Synthesis of 2,4-dihydroxybutyric acid using formaldehyde, methanol, formic acid, sarcosine, and L-malic acid as substrates via an in vitro multi-enzyme system.

[0056] Route 1 synthesizes 2,4-dihydroxybutyric acid using formaldehyde and L-malic acid as substrates. The reaction conditions for the enzyme cascade system are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-malic acid, 2 mM NAD. + 5mM MgCl2, 1mg / ml VhAld, and 2mg / ml Mdh0 were added, with 50mM formaldehyde added every 1 hour for a total of 10 additions. The reaction temperature was 40℃. The amount of 2,4-dihydroxybutyric acid produced was monitored by HPLC analysis.

[0057] Route 2 synthesizes 2,4-dihydroxybutyric acid using methanol and L-malic acid as substrates. The reaction conditions for the enzyme cascade system are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-malic acid, 2 mM NAD. + The reaction was carried out using 5 mM MgCl2, 1 mg / ml VhAld, 2 mg / ml Mdh0, 3 mg / ml BsMdh, and 200 mM methanol at 40 °C. The amount of 2,4-dihydroxybutyric acid produced was monitored by HPLC analysis.

[0058] Pathway 3 synthesizes 2,4-dihydroxybutyric acid using formic acid and L-malic acid as substrates. The reaction conditions of the enzyme cascade system are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-malic acid, 2 mM NAD. + The reaction was catalyzed by adding 5 mM MgCl2, 1 mg / ml VhAld, 2 mg / ml Mdh0, 3 mg / ml PpfdhA, and 200 mM formic acid at 40 °C. The amount of 2,4-dihydroxybutyric acid produced was monitored by HPLC analysis.

[0059] Pathway four synthesizes 2,4-dihydroxybutyric acid using sarcosine and L-malic acid as substrates. The reaction conditions for the enzyme cascade system are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-malic acid, 2 mM NAD. + The reaction was catalyzed by 5 mM MgCl2, 1 mg / ml VhAld, 2 mg / ml Mdh0, 3 mg / ml BsSoxA, and 200 mM sarcosine at 40 °C. The amount of 2,4-dihydroxybutyric acid produced was monitored by HPLC analysis.

[0060] Ultimately, 2,4-dihydroxybutyric acid could be produced using all four methods; detailed HPLC results can be found in [link to HPLC results]. Figure 2The yields of 2,4-dihydroxybutyric acid (2,4-DHA) produced via pathways 1-4 were 11.3 g / L, 3.5 g / L, 2.4 g / L, and 6.3 g / L, respectively. The highest yield was observed when formaldehyde was used directly as the substrate. This was primarily due to the relatively low catalytic efficiency of the enzymes involved in the formaldehyde preparation process, particularly methanol dehydrogenase BsMdh and formaldehyde dehydrogenase PpfdhA, which are the rate-limiting enzymes in their respective pathways.

[0061] In the aforementioned in vitro multi-enzyme catalytic system, the highest yield of 2,4-dihydroxybutyric acid was 11.3 g / L (94 mM). Existing technology CN115948482A synthesizes 2,4-dihydroxybutyric acid from L-lactic acid and formaldehyde using aldolase and dehydrogenase. However, due to the low dehydrogenase activity of the dehydrogenase used for L-lactic acid, the yield only reached 6.54 mM. Therefore, this invention uses L-malic acid as a raw material, increasing the yield by 14 times.

[0062] Example 3: Optimization of component conditions for in vitro multi-enzyme catalytic system

[0063] In the first pathway system, by screening temperature, NAD + The concentrations of *Pseudomonas aeruginosa* oxaloacetate decarboxylase PaOadC and other factors were further optimized to obtain the best reaction conditions for the system. The specific reaction conditions for the enzyme cascade system are as follows:

[0064] (1) When screening at the specified temperature, the conditions for other components are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-malic acid, 2 mM NAD + 5mM MgCl2, 1mg / ml VhAld, 2mg / ml Mdh0, and 50mM formaldehyde were added every 1 hour for a total of 10 additions. The reaction temperatures were 30℃, 35℃, and 40℃.

[0065] (2) Screening for NAD + At the specified concentration, the conditions for other components were as follows: 20 mM HEPES pH 7.5, 0.5 M L-malic acid, 5 mM MgCl2, 1 mg / ml VhAld, 2 mg / ml Mdh0, with 50 mM formaldehyde added every 1 hour for a total of 10 additions. The reaction temperature was 40℃. + The concentrations were 0, 0.5, 1, 2, 5, and 10 mM.

[0066] (3) When screening the concentration of oxaloacetate decarboxylase PaOadC, the other component conditions are as follows: 20 mM HEPES, pH 7.5, 0.5 M L-malic acid, 2 mM NAD. +5 mM MgCl2, 1 mg / ml VhAld, and 2 mg / ml Mdh0 were added, with 50 mM formaldehyde added every 1 hour for a total of 10 additions. The reaction temperature was 40℃. The PaOadC concentrations were 0, 0.5, 1, and 2 mg / ml, respectively. The amount of 2,4-dihydroxybutyric acid generated was finally monitored by HPLC analysis.

[0067] Table 1: DHB production under different conditions.

[0068] Serial Number temperature <![CDATA[NAD + Concentration mM]]> PaOadC concentration (mg / ml) DHB production (g / L) 1 30℃ 2 0 2.9 2 35℃ 2 0 5.8 3 40℃ 2 0 11.3 4 40℃ 0 0 3.1 5 40℃ 0.5 0 7.8 6 40℃ 1 0 9.5 7 40℃ 5 0 11.8 8 40℃ 10 0 11.0 9 40℃ 2 0.5 25 10 40℃ 2 1 38 11 40℃ 2 2 50

[0069] Table 1 shows the DHB yield under different conditions. It can be seen that, compared to 30℃ and 35℃, the in vitro multi-enzyme system produces a higher yield of 2,4-dihydroxybutyrate at 40℃. Simultaneously, the addition of the dehydrogenase cofactor NAD... + It can improve catalytic activity, including the addition of 2-10 mM NAD. + With similar catalytic efficiency, therefore, considering economic factors, adding 2 mM NAD... + The appropriate concentration was determined. Furthermore, the addition of oxaloacetic acid decarboxylase PaOadC significantly increased the yield of 2,4-dihydroxybutyric acid. At PaOadC concentrations of 0-2 mg / ml, the yield of 2,4-dihydroxybutyric acid increased with increasing dosage, reaching a maximum of 50 g / L.

[0070] Example 4: Synthesis of 2,4-dihydroxybutyric acid by microbial fermentation or whole-cell catalysis

[0071] Construction of functional plasmids and bacterial strains:

[0072] The pathway-related plasmids pRSFDuet-1-VhAld-Mdh0, pCDFDuet-1-VhAld-Mdh0, pETDuet-1-VhAld-Mdh0, pACYCDuet-1-VhAld-Mdh0, pCOLADuet-1-VhAld-Mdh0, pTrc99a-VhAld-Mdh0, and pTrc33a-PaOadC were constructed. They were obtained via Gibson assembly using homologous primers.

[0073] Construction of strains: The key gene frmA (NCBIGene ID:944988) in the formaldehyde detoxification system of BL21(DE3) and W3110 strains was knocked out to form functional strains BL21(DE3)△frmA and W3110△frmA.

[0074] Construction of recombinant strains: The five vectors pRSFDuet-1-VhAld-Mdh0, pCDFDuet-1-VhAld-Mdh0, pETDuet-1-VhAld-Mdh0, pACYCDuet-1-VhAld-Mdh0, and pCOLADuet-1-VhAld-Mdh0 were transformed into either BL21(DE3) or BL21(DE3)△frmA strains to form recombinant strains. pTrc99a-VhAld-Mdh0 and pTrc33a-PaOadC were transformed into W3110△frmA to form recombinant strains.

[0075] 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 Tris buffer pH 8.5, 0.5 M L-malic acid, 0.5 mM NAD. + 5 mg / ml MgCl2, 10 mg / ml wet cells, and 50 mM formaldehyde were added every 1 hour for a total of 4 times. The synthesis of 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid) was tested. Results showed that 2,4-dihydroxybutyric acid was produced in all recombinant strain systems. Specific results are shown in Table 2. Among the five Duet vectors, pCOLADuet-1 had the lowest yield. This indicates that the yield of 2,4-dihydroxybutyric acid is related to the copy number of the vector in the strain; a low copy number will affect the yield. Furthermore, knocking out the key gene frmA in the formaldehyde detoxification system significantly increased the yield of 2,4-dihydroxybutyric acid. In the W3110△frmA strain, the introduction of the pathway-related plasmid pTrc99a-VhAld-Mdh0 also produced 2,4-dihydroxybutyric acid. Simultaneously, the introduction of the decarboxylase pTrc33a-PaOadC improved the efficiency of the catalytic system, resulting in a 2.4-fold increase in yield.

[0076] Table 2: Yield of 2,4-dihydroxybutyric acid (g / L) under different conditions

[0077]

[0078] Another approach involved directly culturing the recombinant strain at 37°C using M9 medium. After the OD600 reached 0.4, 0.2 mM IPTG was added, followed by cooling to 30°C and culturing for 3 hours. Then, formaldehyde and L-malic acid were added at a rate of 50 mM / h, repeated 10 times, and the synthesis of 2,4-dihydroxybutyric acid was tested. The results showed that 2,4-dihydroxybutyric acid was produced in all different recombinant strain systems; specific yields are shown in Table 3.

[0079] Table 3: Yield of 2,4-dihydroxybutyric acid under different conditions

[0080]

[0081] Compared to BL21(DE3), the BL21(DE3) strain with the frmA gene knocked out showed a certain increase in the production of 2,4-dihydroxybutyric acid (2,4-dihydroxybutyric acid) in ΔfrmA. Comparing Tables 2 and 3, the yield from the microbial fermentation method was lower, with the highest yield being half that of whole-cell catalysis. The main reason for this is the complex metabolic regulation within microorganisms; the added substrate cannot be fully utilized by the catalytic system, and there are also issues with compatibility with microbial metabolism. Therefore, metabolic engineering is needed to further improve the efficiency of the catalytic system in microbial fermentation.

Claims

1. A method for the biosynthesis of 2,4-dihydroxybutyric acid, characterized in that, Using L-malic acid and formaldehyde as substrates, a multi-enzyme catalytic system is constructed, comprising dehydrogenases, aldolases, or further including oxaloacetate decarboxylase, phosphoenolpyruvate kinase, malate kinase, or pyruvate kinase. The catalytic reaction process is as follows: (1) Dehydrogenase catalyzes the dehydrogenation of L-malate to produce oxaloacetic acid; (2) Oxaloacetic acid is spontaneously decarboxylated or under the action of divalent cations or oxaloacetic acid decarboxylation-related enzymes to generate pyruvate; (3) Aldolase catalyzes the reaction of formaldehyde and pyruvate to produce 2-keto-4-hydroxybutyric acid; (4) Dehydrogenase catalyzes the hydrogenation reaction of 2-keto-4-hydroxybutyric acid to produce 2,4-dihydroxybutyric acid.

2. The method as described in claim 1, characterized in that, This further includes the step of synthesizing formaldehyde using formaldehyde precursor molecules as the initial substrate; Preferably, the formaldehyde precursor is methanol, formic acid, or sarcosine, correspondingly including methanol dehydrogenase, formaldehyde dehydrogenase, or sarcosine oxidase, which generates formaldehyde through an enzymatic catalytic reaction; more specifically, the reaction is carried out according to the following process: methanol dehydrogenase catalyzes methanol to formaldehyde; sarcosine oxidase catalyzes sarcosine to formaldehyde; or formaldehyde dehydrogenase catalyzes formic acid to formaldehyde; further, it also includes directly using fermentation broth containing L-malic acid as the initial raw material.

3. The method as described in claim 1, characterized in that, The oxaloacetate decarboxylation-related enzymes include, but are not limited to, oxaloacetate decarboxylase, phosphoenolpyruvate kinase, malate kinase, or pyruvate kinase.

4. The synthesis method according to claim 1, 2, or 3, characterized in that, The multi-enzyme system synthesizes 2,4-dihydroxybutyric acid in vitro, or through whole-cell or microbial fermentation by recombinant strains expressing various enzymes required for the reaction.

5. The method according to any one of claims 1 to 4, characterized in that, The aldolase mentioned is an aldolase that can catalyze the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyric acid; Specifically, these are either type I or type II aldolases. Examples of type I aldolases include 2-deoxy-3-depentose aldolase (Uniprot entry P39359, SEQ ID No. 1) and 2-dehydro-3-deoxy-D-glucuronide aldolase (Uniprotentry P75682, SEQ ID No. 2). Type II aldolases are proteins in the HpcH family that catalyze the target reaction, specifically 2-dehydro-3-deoxy-L-rhamnolate aldolase (Uniprot entry P76469). To increase solubility, improvements were made based on patent WO2017198717A1, adding an MBP fusion tag. The specific sequence is shown in SEQ ID No. 3 and 4-hydroxy-2-oxovalerate aldolase (Uniprot entry A0A7D8V3B9, SEQ ID No. 4). The dehydrogenase is capable of using L-malic acid and 2-keto-4-hydroxybutyric acid as substrates, specifically L-malic acid dehydrogenase (EC: 1.1.1.37) and its mutants, more specifically a five-site mutant (SEQ ID No. 5) of Escherichia coli L-malic acid dehydrogenase (Uniprot entry P61889). The methanol dehydrogenase is a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase, an NAD-dependent methanol dehydrogenase, or a methanol oxidase. A specific example selected is an NAD-dependent methanol dehydrogenase (Uniprot entry P42327, SEQ ID No. 6). The sarcosine oxidase (EC:1.5.3.1) catalyzes the production of formaldehyde and glycine from sarcosine. A specific example is the sarcosine oxidase of Bacillus (Uniprot entry P40859, SEQ ID No.7). The formaldehyde dehydrogenase (EC: 1.2.1.46) can catalyze the formation of formaldehyde from formic acid. A specific example is the formaldehyde dehydrogenase of *Pseudomonas putida* (Uniprot entry P46154, SEQ ID No. 8). The oxaloacetate decarboxylase (EC: 4.1.1.112) can catalyze the decarboxylation of oxaloacetate to produce pyruvate and carbon dioxide. A specific example is Pseudomonas aeruginosa oxaloacetate decarboxylase (Uniprot entry Q9HUU1, SEQ ID No.9).

6. The method as described in claim 5, characterized in that: The catalytic reaction system is one of the following: (1) 2,4-dihydroxybutyric acid is obtained by catalytic reaction in an in vitro multi-enzyme reaction system containing any of the following buffers: HEPES, Tris, MOPS, Tricine, phosphate to maintain pH, formaldehyde, L-malic acid, MgCl2 or other divalent metal ions, as well as aldolase and dehydrogenase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system. (2) 2,4-dihydroxybutyric acid is obtained by catalytic reaction in an in vitro multi-enzyme reaction system containing any of the following buffers: HEPES, Tris, MOPS, Tricine, phosphate, methanol, L-malic acid, MgCl2 or other divalent metal ions, methanol dehydrogenase, aldolase and dehydrogenase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system. (3) 2,4-dihydroxybutyric acid is obtained by catalytic reaction in an in vitro multi-enzyme reaction system containing any of the following buffers: HEPES, Tris, MOPS, Tricine, phosphate, formic acid, L-malic acid, MgCl2 or other divalent metal ions, formaldehyde dehydrogenase, aldolase and dehydrogenase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system. (4) 2,4-Dihydroxybutyric acid is obtained by catalytic reaction in an in vitro multi-enzyme reaction system containing any of the following buffers: HEPES, Tris, MOPS, Tricine, phosphate to maintain pH, sarcosine, L-malic acid, MgCl2 or other divalent metal ions, sarcosine oxidase, aldolase and dehydrogenase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system.

7. The method as described in claim 6, characterized in that: (1) The reaction system contains 5-50 mM buffer solution, pH between 6 and 10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + And 0.5-2 mg / ml aldolase and 1-3 mg / ml dehydrogenase, 50-500 mM formaldehyde, to catalyze the reaction to obtain 2,4-dihydroxybutyric acid; Preferably, formaldehyde is added to the system via a continuous feeding method; (2) The reaction system contains 5-50 mM buffer solution, pH between 6 and 10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + And 0.5-2 mg / ml aldolase and 1-3 mg / ml dehydrogenase, and 1-4 mg / ml methanol dehydrogenase, 10-500 mM methanol, to catalyze the reaction to obtain 2,4-dihydroxybutyric acid; (3) The reaction system contains 5-50 mM buffer solution, pH between 6 and 10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + And 0.5-2 mg / ml aldolase and 1-3 mg / ml dehydrogenase, and 1-4 mg / ml formaldehyde dehydrogenase, 5-500 mM formic acid, to catalyze the reaction to obtain 2,4-dihydroxybutyric acid; (4) The reaction system contains 5-50 mM buffer solution, pH between 6 and 10, 0.5-10 mM MgCl2 or other divalent metal ions, and 0-10 mM NAD. + And 0.5-2 mg / ml aldolase and 1-3 mg / ml dehydrogenase, and 1-4 mg / ml sarcosine oxidase, 5-500 mM sarcosine, to catalyze the reaction to obtain 2,4-dihydroxybutyric acid; More preferably, based on the above four pathways, 0.5-2 mg / ml oxaloacetate decarboxylase is added to the reaction system to increase the rate of 2,4-dihydroxybutyric acid production.

8. The preparation method according to claim 4, characterized in that, Aldolase and dehydrogenase are expressed in the recombinant strain. Further, methanol dehydrogenase is introduced when methanol is used as the formaldehyde precursor, formaldehyde dehydrogenase is introduced when formic acid is used as the formaldehyde precursor, and sarcosine oxidase is introduced when sarcosine is used as the formaldehyde precursor. Preferably, oxaloacetate decarboxylase is also introduced into the recombinant strain.

9. The preparation method according to claim 8, characterized in that, The recombinant strain is selected from Escherichia coli, Corynebacterium glutamnicum, Bacillus subtilis, lactic acid bacteria, Pseudomonas putida, Methylorubrum extorquens, and Saccharomyces cerevisiae; specifically, the enzyme is expressed on a plasmid or integrated into a chromosome.

10. A recombinant bacterial strain, characterized in that, The recombinant bacteria overexpress dehydrogenase and aldolase in the host cell, and preferably also overexpress oxaloacetate decarboxylase; further, it also overexpresses methanol dehydrogenase or formaldehyde dehydrogenase or sarcosine oxidase in the recombinant strain; and even further, it also overexpresses enzymes related to the L-malate pathway in the recombinant strain. Specifically, the originating strain of the recombinant bacteria is Escherichia coli, more specifically BL21(DE3) or W3110 strain, and preferably the key gene frmA in the formaldehyde detoxification system is knocked out in the recombinant bacteria.

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