Biosynthesis method of L-homoserine

Abstract

The invention belongs to the technical field of biology, and discloses a biosynthesis method of L-homoserine. According to the biosynthesis method, L-homoserine is synthesized by taking L-aspartic acid and formaldehyde as substrates, aldolase and transaminase are adopted, and oxaloacetic acid decarboxylase can be optionally added to improve the efficiency of a catalytic system. The method can be realized through an in-vitro multi-enzyme catalysis system, and can also be completed in a manner of introducing a way into microorganisms. According to the method, methanol, formaldehyde and other carbon resources can be introduced to synthesize the L-homoserine 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 biosynthetic method for L-homoserine, and more specifically a biosynthetic method for L-homoserine using methanol / formaldehyde / sarcosine / formic acid and L-aspartic acid as co-substrates. Background Technology

[0002] L-homoserine is a naturally occurring non-protein amino acid that plays an important role in many fields such as biology, chemical engineering, and medicine. Reports indicate that adding appropriate amounts of homoserine to feed can promote chicken growth similar to threonine (Bryant KI, Dilger RN, Parsons CM, et al. Dietary L-Homoserine SparesThreonine in Chickens. J Nutr, 2009, 139(7):1298-302.). L-homoserine can also be used to synthesize the highly efficient, broad-spectrum, and low-toxicity non-selective herbicide L-glufosinate (Zeiss HJ Enantioselective Synthesis of L-Phosphinothricin from L-Methionine and L-Glutamic Acid Via L-Vinylglycine. Tetrahedron, 1992, 48(38):8263-70.). Furthermore, L-homoserine and its derivatives can be converted into other high-value-added pharmaceutical and chemical intermediates through various enzymatic reactions. Therefore, L-homoserine and its derivatives have important application prospects as pharmaceutical intermediates in pharmacology and physiology.

[0003] Currently, the main methods for producing L-homoserine both domestically and internationally are as follows:

[0004] (1) There are two main chemical methods. One method uses relatively expensive L-methionine as a raw material and methyl iodide or methyl bromide, which have serious biotoxicity, as methylating agents. The amino group is protected by nucleophilic attack, and then the product is obtained by hydrolysis under weakly alkaline conditions. The other method uses γ-butyrolactone and phosphorus tribromide as raw materials. After bromination, α-bromo-γ-butyrolactone is obtained by extraction with ethyl acetate. Then, α-bromo-γ-butyrolactone is hydrolyzed with NaOH and ammoniation is carried out by passing ammonia gas to obtain homoserine. Some of the reagents and byproducts used in the above methods are toxic and are not environmentally friendly.

[0005] (2) Biological methods; including microbial fermentation and bioenzymatic methods. Microbial fermentation has long been widely used in the amino acid production industry and has therefore received widespread attention. Significant progress has been made by deleting the competitive and degradation pathways of L-homoserine in *E. coli* and enhancing the efflux pathway of L-homoserine. For example, Mu et al. achieved a yield of 84.1 g / L in fed-batch fermentation (Mu QX, Zhang SS, Mao XJ, et al. Highly efficient production of L-homoserine in *Escherichia coli* by engineering a redox balance route. *Metab Eng*, 2021, 67:321-9.). However, due to the strong inhibitory effect of homoserine on bacterial growth, increasing yield is difficult.

[0006] In comparison, the enzymatic bioreactor is simpler to operate and monitor, and does not have problems such as bacterial growth inhibition, thus having greater potential for development. Currently, there are reports of using aldolase and transaminase to synthesize D and L-homoserine from alanine and formaldehyde as substrates, with yields reaching 0.4 M (47.6 g / L) (Hernandez K, Bujons J, Joglar J, et al. Combining Aldolases and Transaminases for the Synthesis of 2-Amino-4-hydroxybutanoic Acid. ACS Catalysis, 2017, 7(3): 1707-11.). However, this method requires a high amount of enzymes, with aldolase at 4 mg / ml and transaminase at 20 mg / ml, resulting in high costs.

[0007] In summary, the biosynthesis of L-homoserine has advantages such as being environmentally friendly, but it currently faces problems such as high catalytic efficiency and low cost. Therefore, developing an efficient and economical biosynthesis method for L-homoserine is of great significance and application value. Summary of the Invention

[0008] To address the aforementioned issues, this invention designs a biosynthetic route for L-homoserine, using inexpensive L-aspartic acid as the initial raw material. Under the action of aldolase and transaminase, L-homoserine is condensed with formaldehyde and finally converted into L-homoserine through several steps.

[0009] This invention provides a method for the biosynthesis of L-homoserine, which uses L-aspartic acid as a starting material and, in the presence of a catalytic amount of pyruvate or α-ketoglutarate, converts L-aspartic acid and formaldehyde into L-homoserine under the action of aldolase and transaminase. The entire process releases only one molecule of carbon dioxide and produces no other toxic byproducts, exhibiting high atom economy and environmental friendliness.

[0010] The core approach of this invention is approach one, which uses formaldehyde and L-aspartic acid as raw materials to synthesize L-homoserine. The main steps are as follows: Figure 1 Pathway 1. The reaction is initiated using a catalytic amount of pyruvate. First, aldolase catalyzes the reaction of formaldehyde and pyruvate to produce 2-keto-4-hydroxybutyric acid. 2-keto-4-hydroxybutyric acid then undergoes a transamination reaction with L-aspartic acid under the action of transaminase to produce L-homoserine and oxaloacetate. Oxaloacetate is then decarboxylated spontaneously or by divalent cations or enzymes (oxaloacetate decarboxylase, aldolase, phosphoenolpyruvate kinase, malate kinase, and pyruvate kinase) to produce pyruvate, thus entering the catalytic cycle.

[0011] Furthermore, to initiate the reaction, α-ketoglutarate can be used to replace pyruvate. First, under the action of transaminase, the raw materials L-aspartic acid and α-ketoglutarate undergo a transamination reaction to generate L-glutamic acid and oxaloacetic acid. Oxaloacetic acid is decarboxylated spontaneously or under the action of divalent cations or enzymes (oxaloacetate decarboxylase, aldolase, phosphoenolpyruvate kinase, malic acid kinase, and pyruvate kinase) to generate pyruvate. Subsequently, aldolase catalyzes the reaction of another raw material, formaldehyde, with pyruvate to generate 2-keto-4-hydroxybutyric acid. 2-keto-4-hydroxybutyric acid undergoes a transamination reaction with the raw material L-aspartic acid under the action of transaminase to generate L-homoserine and oxaloacetic acid. Oxaloacetic acid is decarboxylated to generate pyruvate, which then enters the catalytic cycle.

[0012] 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 for the generation of formaldehyde from other precursor molecules. That is, it may also include the following steps: methanol dehydrogenase catalyzes the generation of formaldehyde from methanol (see pathway two); formaldehyde dehydrogenase catalyzes the generation of formaldehyde from formic acid (see pathway three); sarcosine oxidase catalyzes the generation of formaldehyde from sarcosine (see pathway four).

[0013] By combining the above novel pathways, a new biosynthetic pathway for L-homoserine using one-carbon compounds such as methanol, formaldehyde, and formic acid was constructed. This pathway introduces abundant one-carbon compounds into the biosynthesis of L-homoserine, achieving advantages such as reduced production costs, increased molar conversion rate, and atom utilization. The L-homoserine multi-enzyme reaction system and microbial cell factory established based on this novel pathway will have significant application prospects.

[0014] Therefore, this invention provides four biosynthetic pathways for L-homoserine, all of which include aldolase and transaminase. Further derived pathways preferably also include methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase, and preferably also oxaloacetate decarboxylase.

[0015] This invention provides a biosynthetic method for synthesizing L-homoserine from L-aspartic acid and formaldehyde. L-homoserine can be synthesized by catalysis using an in vitro multi-enzyme system containing various enzymes required for the reaction, or by whole-cell or microbial fermentation using recombinant strains expressing these enzymes.

[0016] Furthermore, by overexpressing aldolase and transaminase genes in the host, L-homoserine is synthesized through whole-cell catalysis or microbial fermentation after the addition of substrate.

[0017] More preferably, expression plasmids for aldolase and transaminase 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.

[0018] Furthermore, aldolase and transaminase were combined in vitro and reacted with the substrate in a one-pot reaction to synthesize L-homoserine.

[0019] Preferably, the reaction system in pathway one contains the following components: 5-50 mM buffer solution, pH 6-10, 0.1-2 mM pyridoxal phosphate (PLP), 0.5-10 mM MgCl2 or other divalent metal ions, 0.5-10 mM pyruvate or ketoglutarate, 20-30 μM aldolase and 20-50 μM transaminase, and 5-200 mM formaldehyde, to catalyze the reaction and obtain L-homoserine.

[0020] Preferably, formaldehyde is added in batches at a concentration of 10 mM, or added slowly in a fed-batch manner, which improves the synthesis of L-homoserine using an in vitro catalytic system.

[0021] Preferably, the reaction system in pathway two contains the following components: 5-50 mM buffer solution, pH 6-10, 0.1-2 mM pyridoxal phosphate (PLP), 0.5-10 mM MgCl2 or other divalent metal ions, 0.5-10 mM pyruvate or ketoglutarate, 20-50 μM methanol dehydrogenase, 20-30 μM aldolase, 20-50 μM transaminase, and 5-500 mM methanol, to catalyze the reaction and obtain L-homoserine.

[0022] Preferably, the reaction system in pathway three contains the following components: 5-50 mM buffer solution, pH 6-10, 0.1-2 mM pyridoxal phosphate (PLP), 0.5-10 mM MgCl2 or other divalent metal ions, 0.5-10 mM pyruvate or ketoglutarate, 20-50 μM formaldehyde dehydrogenase, 20-30 μM aldolase, 20-50 μM transaminase, and 5-500 mM formic acid, to catalyze the reaction and obtain L-homoserine.

[0023] Preferably, the reaction system in pathway four is supplemented with the following components: 5-50 mM buffer solution, pH between 6 and 10, 0.1-2 mM pyridoxal phosphate (PLP), 0.5-10 mM MgCl2 or other divalent metal ions, 0.5-10 mM pyruvate or ketoglutarate, 20-50 μM sarcosine oxidase, 20-30 μM aldolase, 20-50 μM transaminase, and 5-500 mM sarcosine, to catalyze the reaction and obtain L-homoserine.

[0024] More preferably, by adding 0.5-2 mg / ml oxaloacetate decarboxylase to the reaction system based on the above four pathways, the L-homoserine production rate can be further increased.

[0025] This invention further provides a genetically engineered recombinant strain for producing L-homoserine. The recombinant strain overexpresses transaminases and aldolases in host cells, thereby synthesizing L-homoserine. 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. More preferably, the recombinant strain overexpresses oxaloacetate decarboxylase to increase the oxaloacetate decarboxylation rate. Simultaneously, it may also include overexpression of enzymes related to the L-aspartate pathway in the recombinant strain, i.e., introducing the novel pathway of this invention into a high-yield L-aspartate engineered strain, with the high-yield strain providing the ability to synthesize L-aspartate.

[0026] Preferably, it is selected from Escherichia coli, Corynebacterium glutamnicum, Bacillus subtilis, lactic acid bacteria, Pseudomonas putida, Methylorubrum extorquens, and Saccharomyces cerevisiae;

[0027] The encoding gene is introduced and expressed by constructing it on a plasmid, or by integrating it into the chromosome of a recombinant strain and expressing it.

[0028] 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-hydroxybutyrate; specifically, it is a type I aldolase or a type II aldolase; specific examples of type I aldolases are 2-deoxy-3-depentanolaldolase (Uniprot entry P39359, SEQ ID No. 1) and 2-dehydro-3-deoxy-D-glucuronide aldolase (Uniprot entry P75682, SEQ ID No. 2); the type II aldolase is a protein in the HpcH family that can catalyze the target reaction, a specific example being 2-dehydro-3-deoxy-L-rhamnolate aldolase (Uniprot entry P76469). To increase solubility, it is improved according to WO2017198717A1 by adding an MBP fusion tag, the specific sequence of which can be found in SEQ ID No. 3 and 4-hydroxy-2-oxovalerate aldolase (Uniprot entry P75682, SEQ ID No. 2). A0A254U9J8, SEQ ID No. 4).

[0029] In one embodiment of the present invention, the transaminase is a transaminase that can use L-aspartic acid and 2-keto-4-hydroxybutyric acid as substrates, and can also use L-aspartic acid and α-ketoglutarate as substrates, specifically aspartate transaminase (EC: 2.6.1.1), and more specifically Escherichia coli aspartate transaminase (Uniprot entry P00509, SEQ ID No. 5).

[0030] 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).

[0031] 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, and a specific example is the sarcosine oxidase of Bacillus subtilis (Uniprot entry P40859, SEQ ID No. 7).

[0032] In one embodiment of the present invention, 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).

[0033] 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).

[0034] This invention provides a novel biosynthesis method for L-homoserine. The method involves a basic pathway comprising transaminase and aldolase elements. Through an in vitro multi-enzyme reaction system or by overexpressing two enzyme elements in recombinant microorganisms, a novel pathway for the synthesis of L-homoserine from formaldehyde and L-aspartic acid has been successfully constructed using both methods. Based on this basic pathway, it can further include enzyme elements catalyzing the synthesis of formaldehyde: methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase, etc.; through an in vitro multi-enzyme reaction system or by overexpressing three basic enzyme elements—methanol dehydrogenase, sarcosine oxidase, or formaldehyde dehydrogenase—as well as transaminase and aldolase, a pathway for the conversion of methanol, sarcosine, or formic acid to L-homoserine has been successfully constructed. Furthermore, the introduction of an oxaloacetate decarboxylase element can accelerate the decarboxylation process of oxaloacetate, making the pathway faster and more efficient.

[0035] In summary, this invention introduces high-energy-density, low-cost one-carbon compounds such as methanol, formaldehyde, and formic acid into the synthesis pathway of L-homoserine, resulting in low cost and significant application prospects. Attached Figure Description

[0036] Figure 1 The biosynthetic pathway of L-homoserine.

[0037] Figure 2 HPLC chromatogram of L-homoserine synthesized from formaldehyde and L-aspartic acid using an in vitro multi-enzyme system. Detailed Implementation

[0038] 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.

[0039] Unless otherwise specified, the experimental or measurement methods used in this invention shall be performed as follows:

[0040] Amino acid determination: After the enzymatic reaction, an equal volume of 20% (m / v) trichloroacetic acid solution was added to denature the protein. The protein was centrifuged and filtered through a 0.22 μm filter. HPLC analysis was performed using an Agilent C18 column (5 μm, 5.0 × 240 mm). Analysis was performed using the OPA pre-column derivatization method and reagents provided by Agilent. Mobile phase A consisted of 10 mM Na₂HPO₄ and 10 mM Na₂B₄O₇, pH 8.2; mobile phase B consisted of acetonitrile:methanol:water (45:45:10, v:v:v). The three derivatization reagents were: injection diluent of 100 mL mobile phase A and 0.4 mL concentrated H₃PO₄; OPA reagent: 10 mg / mL, dissolved in 0.4 M borate buffer and 3-mercaptopropionic acid; borate buffer: prepared with 0.4 M boric acid and adjusted to pH 10.4 with NaOH. The flow rate was 1.5 mL / min, detection was performed at 338 nm, and the column temperature was 30 °C. Elution conditions: Gradient elution, mobile phase (B) changes from 0 to 100% within 0-14 min, 100% B within 14-16 min, and 100% A within 16.2-20 min.

[0041] The culture medium used in this invention is as follows:

[0042] (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.

[0043] (2) LB medium (1L): 10g peptone, 10g sodium chloride, 5g yeast extract, pH 7.0.

[0044] Protein overexpression culture conditions:

[0045] 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.

[0046] 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.

[0047] Example 1: Screening of key enzymes in the biosynthetic pathway of L-homoserine and construction of plasmids

[0048] This invention introduces one-carbon compounds such as methanol / formaldehyde into the L-homoserine synthesis pathway. The basic synthetic route is as follows: Figure 1 Pathway 1: Using formaldehyde and L-aspartic acid as substrates, L-homoserine is synthesized by transaminase and aldolase catalysis.

[0049] Aldolases are aldolases that catalyze the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyrate; specifically, they are either type I or type II aldolases. Specific examples of type I aldolases are 2-deoxy-3-depentosealdehyde aldolase (Uniprot entry P39359, SEQ ID No. 1), named EcYjhH, and 2-dehydro-3-deoxy-D-glucuronide aldolase (Uniprot entry P75682, SEQ ID No. 2), named EcYagE. The aforementioned class II aldolases are proteins in the HpcH family that catalyze the target reaction. A specific example is 2-dehydro-3-deoxy-L-rhamnolate aldolase (Uniprotentry P76469). To increase solubility, it is improved with reference to patent WO2017198717A1 by adding an MBP fusion tag, the specific sequence of which can be found in SEQ ID No. 3, named MBP-YfaU, and 4-hydroxy-2-oxovalerate aldolase (Uniprot entry A0A254U9J8, SEQ ID No. 4), named AnAld. The transaminase is a transaminase that can use L-aspartic acid and 2-keto-4-hydroxybutyric acid as substrates, as well as L-aspartic acid and α-ketoglutarate as substrates. Specifically, it is aspartate transaminase (EC: 2.6.1.1), and more specifically, it is Escherichia coli aspartate transaminase (Uniprot entry P00509, SEQ ID No. 5), named EcAspC.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] (1) Plasmid construction and preparation of expression strains

[0055] A series of target genes from the sequence listing were constructed into the pET16b vector by Genewiz using whole-genome synthesis. Codon optimization was performed using the E. coli system, and the vector was transformed into the BL21(DE3) expression strain to obtain the recombinant strain.

[0056] (2) Bacterial culture and induced expression

[0057] 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.

[0058] 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.

[0059] (3) Protein purification

[0060] Proteins were purified using His-tag chelating resin and magnetic beads. The obtained proteins were then treated with a desalting column to remove excess imidazole, followed by activity testing or cryopreservation at -80°C.

[0061] Example 2: Synthesis of L-homoserine using formaldehyde, methanol, formic acid, sarcosine, and L-aspartic acid as substrates via an in vitro multi-enzyme system.

[0062] Route 1 synthesizes L-homoserine using formaldehyde and L-aspartic acid as substrates. The reaction conditions of the enzyme cascade system are as follows: 20 mM HEPES pH 7.5, 100 mM L-aspartic acid, 0.2 mM PLP, 5 mM MgCl2, 10 mM pyruvate, and 20 μM MBP-YfaU or 30 μM MAnAld (0.9 mg / ml) or 30 μM MEcYjhH or 30 μM MEcYagE and 30 μM MEcAspC (1.4 mg / ml). 20 mM formaldehyde is added every 3 hours for a total of 5 additions. The amount of L-homoserine produced is monitored by HPLC analysis.

[0063] Route 2 synthesized L-homoserine using methanol and L-aspartic acid as substrates. The reaction conditions of the enzyme cascade system were as follows: 20 mM HEPES pH 7.5, 100 mM L-aspartic acid, 0.2 mM PLP, 5 mM MgCl2, 10 mM pyruvate, 30 μM AmAld (0.9 mg / ml) and 30 μM EcAspC (1.4 mg / ml), 30 μM BsMdh, 200 mM methanol, and the reaction was carried out overnight at room temperature. The amount of L-homoserine produced was monitored by HPLC analysis.

[0064] Pathway 3 synthesizes L-homoserine using formic acid and L-aspartic acid as substrates. The reaction conditions of the enzyme cascade system are as follows: 20 mM HEPES pH 7.5, 100 mM L-aspartic acid, 0.2 mM PLP, 5 mM MgCl2, 10 mM pyruvate, 30 μM MAnAld (0.9 mg / ml) and 30 μM MEcAspC (1.4 mg / ml), 30 μM PpfdhA, 200 mM formic acid, reacted overnight at room temperature, and the amount of L-homoserine produced was monitored by HPLC analysis.

[0065] Pathway 4 synthesizes L-homoserine using sarcosine and L-aspartic acid as substrates. The reaction conditions of the enzyme cascade system are as follows: 20 mM HEPES pH 7.5, 100 mM L-aspartic acid, 0.2 mM PLP, 5 mM MgCl2, 10 mM pyruvate, 30 μM MAnAld (0.9 mg / ml) and 30 μM MEcAspC (1.4 mg / ml), 30 μM MBsSoxA, 200 mM sarcosine, reacted overnight at room temperature, and the amount of L-homoserine produced was monitored by HPLC analysis.

[0066] Based on the above-described pathway conditions, 30 μMPa OadC was added. Ultimately, L-homoserine was produced using all four pathways. Specific yields are shown in Table 1.

[0067] Table 1: L-homoserine yield under different conditions.

[0068]

[0069]

[0070] Example 3: Optimization of formaldehyde addition rate in pathway one

[0071] In the first pathway, aldolase requires pyruvate as a substrate, and pyruvate requires oxaloacetate produced by transaminase, which is then decarboxylated. Therefore, the pyruvate supply rate and the formaldehyde addition rate need to be balanced. If formaldehyde is added too quickly, it will accumulate and cause protein toxicity. In Example 2, the formaldehyde addition rate was set at 20 mM formaldehyde every 3 hours, for a total of 5 additions. Based on this, the reaction conditions were adjusted as follows: 20 mM HEPES pH 7.5, 200 mM L-aspartic acid, 0.2 mM PLP, 5 mM MgCl2, 10 mM pyruvate, and 20 μM MBP-YfaU or 30 μM MAnAld (0.9 mg / ml) and 30 μM MEcAspC (1.4 mg / ml) and 30 μM PaOadC, with 20 mM formaldehyde added every 5 hours, for a total of 10 additions. The amount of L-homoserine generated was monitored by HPLC analysis. See the specific HPLC chromatogram below. Figure 2 L-Aspartic acid was almost completely converted. In the AnAld aldolase group, the L-homoserine yield reached 170 mM (20 g / L), and in the MBP-YfaU group it was 158 mM. Both groups produced a small amount of L-alanine byproduct.

[0072] Example 4: Synthesis of L-homoserine by microbial fermentation or whole-cell catalysis

[0073] Construction of functional plasmids and strains: Construction pathway-related plasmids pRSFDuet-1-AnAld-EcAspC, pCDFDuet-1-AnAld-EcAspC, pETDuet-1-AnAld-EcAspC, pACYCDuet-1-AnAld-EcAspC, pCOLADuet-1

[0074] -AnAld-EcAspC, pTrc99a-AnAld-EcAspC, pTrc33a-PaOadC. These were obtained by designing homologous primers and assembling them using the Gibson method.

[0075] 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.

[0076] Construction of recombinant strains: The five vectors pRSFDuet-1-AnAld-EcAspC, pCDFDuet-1-AnAld-EcAspC, pETDuet-1-AnAld-EcAspC, pACYCDuet-1-AnAld-EcAspC, and pCOLADuet-1-AnAld-EcAspC were transformed into either BL21(DE3) or BL21(DE3)△frmA strains to form recombinant strains. The vectors pTrc99a-AnAld-EcAspC and pTrc33a-PaOadC were transformed into W3110△frmA to form recombinant strains.

[0077] Further, the recombinant strain was seed cultured, and whole cells were obtained through an induced expression process, followed by enzymatic reactions. The whole-cell reaction system was as follows: 20 mM HEPES pH 7.5, 100 mM L-aspartic acid, 0.2 mM PLP, 5 mM MgCl2, 10 mM pyruvate, 10 mg / ml wet cells, and 10 mM formaldehyde added every 5 hours for a total of 10 times. The L-homoserine synthesis was tested. The results showed that L-homoserine was produced in all different recombinant strain systems. Specific results are shown in Table 2. Among the five Duet vectors, pRSFDuet-1 had the highest yield, while pCOLADuet-1 had the lowest yield, indicating that L-homoserine production is related to the copy number of the vector in the strain; a higher copy number of pRSFDuet-1 resulted in higher production. In addition, knocking out the key gene frmA in the formaldehyde detoxification system significantly increased L-homoserine production. In strain W3110△frmA, the introduction of pathway-related plasmid pTrc99a-AnAld-EcAspC also enabled the production of L-homoserine. At the same time, the introduction of decarboxylase pTrc33a-PaOadC improved the efficiency of the catalytic system, resulting in a 2.8-fold increase in yield.

[0078] Table 2: L-homoserine yield under different conditions.

[0079]

[0080]

[0081] 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-aspartic acid were added at 20 mM levels every 6 hours for a total of 5 additions, and the L-homoserine synthesis was tested. The results showed that L-homoserine was produced in all different recombinant strain systems; specific yields are shown in Table 3.

[0082] Table 3: L-homoserine yield under different conditions.

[0083]

[0084] Comparing Tables 2 and 3, the yield of the microbial fermentation method is relatively low. This is mainly because there is complex metabolic regulation in the microorganisms, the added substrates cannot be fully utilized by the catalytic system, and there are also problems such as incompatibility with microbial metabolism. Therefore, metabolic engineering is needed to further improve the efficiency of the catalytic system in the microbial fermentation process.

Claims

1. A method for the biosynthesis of L-homoserine, characterized in that, Using L-aspartic acid and formaldehyde as substrates, a multi-enzyme catalytic system is constructed, comprising transaminases, aldolases, or further including oxaloacetate decarboxylase, phosphoenolpyruvate kinase, malic acid kinase, or pyruvate kinase. The catalytic reaction process is as follows: (1) Transaminase catalyzes the transamination of L-aspartic acid 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) Transaminase catalyzes the reaction of L-aspartic acid with 2-keto-4-hydroxybutyric acid to generate L-homoserine.

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 as described in claim 3, characterized in that, The multi-enzyme system synthesizes L-homoserine through in vitro catalysis, or through the synthesis of L-homoserine by expressing recombinant strains of various enzymes required for the reaction via whole-cell or microbial fermentation.

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, it refers to either type I or type II aldolases; specific examples of type I aldolases are 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). The class II aldolases are proteins in the HpcH family that can catalyze the target reaction. Specific examples include 2-dehydro-3-deoxy-L-rhamnolate aldolase (Uniprot entry P76469), or further enhanced with an MBP fusion tag, the specific sequences of which can be found in SEQ ID No. 3 and 4-hydroxy-2-oxovalerate aldolase (Uniprot entry A0A254U9J8, SEQ ID No. 4). The transaminase is a transaminase that can use L-aspartic acid and 2-keto-4-hydroxybutyric acid as substrates, as well as L-aspartic acid and α-ketoglutarate as substrates, specifically aspartate transaminase (EC: 2.6.1.1), and more specifically Escherichia coli aspartate transaminase (Uniprot entry P00509, SEQ ID No. 5); 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 4, characterized in that: (1) L-homoserine is obtained by catalyzing a multi-enzyme reaction system containing any of the following: HEPES, Tris, MOPS, Tricine, phosphate to maintain pH, formaldehyde, pyridoxal phosphate PLP, MgCl2 or other divalent metal ions, pyruvate or ketoglutarate, as well as aldolase and transaminase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system. (2) L-homoserine 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, methanol, pyridoxal phosphate PLP, MgCl2 or other divalent metal ions, pyruvate or ketoglutarate, as well as methanol dehydrogenase, aldolase and transaminase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system. (3) L-homoserine is obtained by catalyzing a reaction in an in vitro multi-enzyme reaction system containing any of the following: HEPES, Tris, MOPS, Tricine, phosphate to maintain pH, formic acid, pyridoxal phosphate PLP, MgCl2 or other divalent metal ions, pyruvate or ketoglutarate, formaldehyde dehydrogenase, aldolase and transaminase. Preferably, oxaloacetate decarboxylase is added to improve the efficiency of the entire catalytic system. (4) L-homoserine is obtained by catalyzing a multi-enzyme reaction system containing any of the following: HEPES, Tris, MOPS, Tricine, phosphate to maintain pH, sarcosine, pyridoxal phosphate PLP, MgCl2 or other divalent metal ions, pyruvate or ketoglutarate, as well as sarcosine oxidase, aldolase and transaminase. 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 5-50mM buffer solution, pH between 6 and 10, 0.1-2mM pyridoxal phosphate (PLP), 0.5-10mM MgCl2 or other divalent metal ions, 0.5-10mM pyruvate or ketoglutarate, 20-30µM aldolase and 20-50µM transaminase, and 5-200mM formaldehyde to catalyze the reaction to obtain L-homoserine; More preferably, formaldehyde is added to the system in a continuous feeding manner; Preferably, 0.5-2 mg / ml oxaloacetate decarboxylase is added to the reaction system to increase the L-homoserine production rate; (2) The reaction system contains 5-50mM buffer solution, pH between 6 and 10, 0.1-2mM pyridoxal phosphate (PLP), 0.5-10mM MgCl2 or other divalent metal ions, 0.5-10mM pyruvate or ketoglutarate, 20-50µM methanol dehydrogenase, 20-30µM aldolase and 20-50µM transaminase, and 5-500mM methanol to catalyze the reaction to obtain L-homoserine; Preferably, 0.5-2 mg / ml oxaloacetate decarboxylase is added to the reaction system to increase the L-homoserine production rate; (3) The reaction system contains 5-50mM buffer solution, pH between 6 and 10, 0.1-2mM pyridoxal phosphate (PLP), 0.5-10mM MgCl2 or other divalent metal ions, 0.5-10mM pyruvate or ketoglutarate, 20-50µM formaldehyde dehydrogenase, 20-30µM aldolase and 20-50µM transaminase, and 5-500mM formic acid, to catalyze the reaction and obtain L-homoserine; Preferably, 0.5-2 mg / ml oxaloacetate decarboxylase is added to the reaction system to increase the L-homoserine production rate; (4) The reaction system contains 5-50mM buffer solution, pH between 6 and 10, 0.1-2mM pyridoxal phosphate (PLP), 0.5-10mM MgCl2 or other divalent metal ions, 0.5-10mM pyruvate or ketoglutarate, 20-50µM sarcosine oxidase, 20-30µM aldolase and 20-50µM transaminase, and 5-500mM sarcosine to catalyze the reaction to obtain L-homoserine; Preferably, 0.5-2 mg / ml oxaloacetate decarboxylase is added to the reaction system to increase the L-homoserine production rate.

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

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

10. A recombinant strain of L-homoserine, characterized in that, It contains genes encoding transaminases, aldolases, or further including oxaloacetate decarboxylase, phosphoenolpyruvate kinase, malate kinase, or pyruvate kinase that can be expressed. More preferably, the recombinant strain also includes overexpression of methanol dehydrogenase, formaldehyde dehydrogenase, or sarcosine oxidase to convert compounds such as methanol, formic acid, or sarcosine into formaldehyde. Selected from Escherichia coli, Corynebacterium glutamnicum, Bacillus subtilis, Lactic acid bacteria, Pseudomonas putida, Methylorubrum extorquens, and Saccharomyces cerevisiae; The encoding gene is introduced and expressed by constructing it on a plasmid, or by integrating it into the chromosome of a recombinant strain and expressing it.

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