A method for the biosynthesis of 1,3-propanediol

Abstract

The application provides a biosynthesis method of 1,3-propanediol, which comprises the following steps: (i) converting lactic acid into pyruvic acid; (ii) condensing the generated pyruvic acid with formaldehyde to generate 2-keto-4-hydroxybutyric acid; (iii) decarboxylating the generated 2-keto-4-hydroxybutyric acid to generate 3-hydroxypropionaldehyde; and (iv) reducing the generated 3-hydroxypropionaldehyde to generate 1,3-propanediol. The method has the advantages of mild reaction condition, high atom economy, release of only one molecule of CO2, and no toxic by-products. The method provided by the application has the advantages of low cost, high stability, easy amplification, green and low carbon, and is suitable for industrialized production.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, specifically to a method for the biosynthesis of 1,3-propanediol, and more specifically to a method for the biosynthesis of 1,3-propanediol based on the self-balancing of NAD cofactor and using lactic acid and formaldehyde as substrates. Background Technology

[0002] 1,3-Propanediol (1,3-PDO) is an important bulk chemical intermediate and bio-based platform compound, widely used in polypropylene terephthalate (PTT) fibers, polyurethanes, pharmaceutical intermediates, cosmetics, and high-performance materials. It holds an irreplaceable core position in high-end textiles, automobiles, electronics, and daily chemical industries. The global 1,3-PDO market is steadily growing, with broad prospects for industrial development. Currently, global industrial production of 1,3-PDO mainly relies on petroleum-based chemical synthesis routes. While the technology is mature and costs are relatively controllable, it suffers from significant drawbacks, including non-renewable raw materials, harsh reaction conditions, high energy consumption, large emissions of waste, and high carbon intensity, which are seriously inconsistent with the trends of green, low-carbon, and sustainable development. In contrast, biosynthesis uses renewable resources as raw materials, has mild reaction conditions, is environmentally friendly, and has a low carbon footprint. It is a core direction for replacing traditional chemical methods and achieving green and low-carbon manufacturing, attracting considerable attention from both academia and industry.

[0003] However, existing 1,3-PDO biosynthesis technologies still face a series of key bottlenecks: mainstream routes are highly dependent on high-purity glycerol or grain sugar carbon sources, creating a contradiction of "competing with grain"; strains have poor substrate tolerance, complex metabolic pathways, and serious byproduct accumulation, resulting in low conversion efficiency; the process relies on exogenous coenzymes or specific functional enzymes, leading to high costs; separation and purification costs are high, and downstream extraction and refining account for a high proportion of the total cost, making it uneconomical and difficult to simultaneously achieve high efficiency, low cost, broad-spectrum raw materials, and low carbon emissions, urgently requiring breakthroughs in original innovation.

[0004] To overcome the raw material bottleneck, synthetic routes using inexpensive and renewable small molecules derived from C1 resources and agricultural, forestry, and industrial waste as substrates have become an important research direction. In 2019, Wang et al. reported a biosynthetic route for 1,3-PDO using methanol as a substrate, constructing an artificial pathway to achieve synthesis through pyruvate-dependent aldolase-mediated formaldehyde assimilation. Although this route verified the feasibility of utilizing C1 raw materials, methanol must first undergo dehydrogenation to generate formaldehyde before participating in the reaction, making the process cumbersome, resulting in serious accumulation of byproducts (ethanol, formic acid, etc.), low conversion efficiency, poor system stability, and susceptibility to imbalance; thus, it is difficult to achieve large-scale, low-cost industrial applications.

[0005] In summary, existing 1,3-PDO biosynthesis technologies generally suffer from problems such as reliance on raw materials and coenzymes, high costs, and insufficient atom economy, making it difficult to support large-scale green manufacturing. Therefore, developing novel 1,3-PDO biosynthesis technologies that use inexpensive, renewable small molecules as raw materials, require no exogenous coenzymes, have high atom economy, and controllable costs is of great significance for promoting the upgrading of the bio-manufacturing industry and enhancing the competitiveness of my country's bio-based materials industry chain. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing 1,3-propanediol biosynthesis technologies, such as reliance on exogenous coenzymes, poor system stability, low efficiency of key conversion steps, and high cost. In particular, it addresses the technical bottleneck of low methanol-to-formaldehyde conversion efficiency in existing C1 conversion routes, which is prone to becoming the rate-limiting step. This invention provides a 1,3-propanediol biosynthesis method based on NAD cofactor self-balancing and using lactic acid and formaldehyde as substrates, to achieve low-cost, high-stability, easy-to-scale, green and low-carbon large-scale production.

[0007] The first aspect of this invention provides a method comprising the following enzymatic catalytic steps: (i) Converting lactic acid to pyruvate; (ii) The generated pyruvate condenses with formaldehyde to form 2-keto-4-hydroxybutyric acid; (iii) The generated 2-keto-4-hydroxybutyric acid undergoes decarboxylation to generate 3-hydroxypropanal; (iv) The generated 3-hydroxypropanal is reduced to 1,3-propanediol.

[0008] In some embodiments, in step (i), the conversion of lactate to pyruvate is achieved using a lactate dehydrogenase classified under EC 1.1.1.-. In some embodiments, the lactate dehydrogenase includes one or more of L-lactate dehydrogenase (EC: 1.1.1.27) and its variants, and D-lactate dehydrogenase (EC: 1.1.1.28) and its variants. In some embodiments, the lactate dehydrogenase includes one or more of L-lactate dehydrogenase or its variants from *Lactobacillus pentosus*, and L-lactate dehydrogenase or its variants from *Mycoplasma synoviae*. In some embodiments, the L-lactate dehydrogenase from *Lactobacillus pentosus* comprises an amino acid sequence as shown in SEQ ID No: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it, and / or the L-lactate dehydrogenase from *Mycoplasma synoviae* comprises an amino acid sequence as shown in SEQ ID No: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0009] In some embodiments, in step (ii), an aldolase classified under EC 4.1.2.- is used to condense the generated pyruvate with formaldehyde to form 2-keto-4-hydroxybutyrate. In some embodiments, the aldolase includes one or more of class I aldolases or variants thereof, and type II aldolases or variants thereof. In some embodiments, the aldolase includes one or more of type II aldolases or variants thereof from the genus *Porcinidia*. In some embodiments, the type II aldolase from the genus *Porcinidia* comprises an amino acid sequence as shown in SEQ ID No: 3 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0010] In some embodiments, in step (iii), the generated 2-keto-4-hydroxybutyrate is decarboxylated to 3-hydroxypropionaldehyde using a decarboxylase classified under EC 4.1.1. In some embodiments, the decarboxylase comprises one or more keto acid decarboxylases (EC 4.1.1.1) or variants thereof. In some embodiments, the decarboxylase comprises one or more keto acid decarboxylases derived from Lactococcus lactis or variants thereof. In some embodiments, the Lactococcus lactis keto acid decarboxylase variant comprises an amino acid sequence as shown in SEQ ID No: 4 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0011] In some embodiments, in step (iv), the generated 3-hydroxypropionaldehyde is reduced to 1,3-propanediol using a reductase classified as EC 1.1.1.-. In some embodiments, the reductase comprises one or more of 1,3-propanediol dehydrogenase (EC 1.1.1.202) or variants thereof. In some embodiments, the reductase comprises one or more of 1,3-propanediol dehydrogenase from Klebsiella pneumoniae or variants thereof. In some embodiments, the 1,3-propanediol dehydrogenase from Klebsiella pneumoniae comprises the amino acid sequence shown in SEQ ID No: 5 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0012] In some embodiments, the method employs an in vitro multi-enzyme catalytic system.

[0013] In some embodiments, the in vitro multi-enzyme catalytic system comprises: 0-10 mM (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM) divalent metal ions, 0-2 mM (e.g., 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 mM) thiamine pyrophosphate (TPP), and 0-2 mM (e.g., 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 mM) NAD+. + 1-3 mg / mL (e.g., 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3.0 mg / mL) lactate dehydrogenase; 0.5-2 mg / mL (e.g., 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, or 2.00 mg / mL) aldolase; 0.5-3 mg / mL (e.g., 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, or 3.00 mg / mL) decarboxylase; 0.5-3 mg / mL (e.g., 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75 or 3.00 mg / mL) reductase, 20-1000 mM (e.g., 20, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM) lactic acid, 20-500 mM (e.g., 20, 70, 120, 170, 220, 270, 320, 370, 420, 470 or 500 mM) formaldehyde.

[0014] In some embodiments, the in vitro multi-enzyme catalytic system comprises: 2-7 mM (e.g., 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 mM) divalent metal ions, 0.1-1 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM) thiamine pyrophosphate (TPP), and 0.25-0.75 (e.g., 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, or 0.75 mM) mM NAD. +1.5-3 mg / mL (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg / mL) lactate dehydrogenase; 0.5-1.5 mg / mL (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mg / mL) aldolase; 1.5-3 mg / mL (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg / mL) decarboxylase; 1.5-3 mg / mL (e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 mg / mL) reductase, 50-200 mM (e.g., 50, 75, 100, 125, 150, 175 or 200 mM) lactic acid, 50-200 mM (e.g., 50, 75, 100, 125, 150, 175 or 200 mM) formaldehyde.

[0015] In some embodiments, the divalent metal ion includes Mg. 2+ In some embodiments, the Mg 2+ It comes from magnesium sulfate or magnesium chloride.

[0016] In some embodiments, the in vitro multi-enzyme catalytic system comprises: 2-5 mg / mL (e.g., 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / mL) lactate dehydrogenase, 1-3 mg / mL (e.g., 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg / mL) aldolase, 2-5 mg / mL (e.g., 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / mL) decarboxylase, and 2-5 mg / mL (e.g., 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / mL) decarboxylase. Reductase (mg / mL), 20-1000 mM (e.g., 20, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM), lactic acid, and 20-500 mM (e.g., 20, 70, 120, 170, 220, 270, 320, 370, 420, 470 or 500 mM) formaldehyde.

[0017] In some embodiments, the lactate dehydrogenase, aldolase, decarboxylase, and reductase in the in vitro multi-enzyme catalytic system are lactate dehydrogenases treated with cofactor completion; the aldolases are aldolases treated with cofactor completion; the decarboxylases are decarboxylases treated with cofactor completion; and the reductases are reductases treated with cofactor completion. The cofactor completion treatment includes mixing the enzyme and cofactor and incubating (e.g., incubating at 4 °C for 2-4 h) to ensure sufficient binding between the cofactor and the enzyme.

[0018] In some embodiments, the lactate dehydrogenase treated with cofactor supplementation is NAD+-treated. + Completed lactate dehydrogenase. In some embodiments, NAD+ is used. + The lactate dehydrogenase that completes the process is to convert lactate NAD+ into NAD+. + The aldolase is obtained by incubation after mixing with dehydrogenase in a molar ratio of (20:1) to (5:1) (e.g., 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, preferably 20:1). The aldolase treated with cofactor completion is obtained by incubation with Mg... 2+ Aldolase with complete treatment. In some embodiments, it is treated with Mg... 2+ The aldolase used in the completion treatment is Mg 2 + It is obtained by incubation after mixing with aldolase in a molar ratio of (20:1) to (5:1) (e.g., 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, preferably 20:1), wherein Mg 2+ The enzyme is derived from magnesium sulfate or magnesium chloride. The cofactor-completed decarboxylase is a TPP-completed decarboxylase. In some embodiments, the TPP-completed decarboxylase is obtained by mixing TPP and the decarboxylase in a molar ratio of (20:1) to (5:1) (e.g., 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, preferably 20:1) and then incubating. The cofactor-completed reductase is a NADH-completed reductase. In some embodiments, the NADH-completed reductase is obtained by mixing NADH and reductase in a molar ratio of (20:1) to (5:1) (e.g., 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, preferably 20:1) and then incubating.

[0019] In some embodiments, the in vitro multi-enzyme catalytic system further includes a buffer system selected from any one of HEPES, Tris, MOPS, Tricine, or phosphate buffer, with a concentration of 5-50 mM (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM) and a pH of 5-10 (e.g., 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0).

[0020] In some embodiments, the reaction temperature of the in vitro multi-enzyme catalytic system is 25-45°C (e.g., 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45°C) and / or the reaction time is 1-72 h (e.g., 1, 2, 4, 8, 12, 24, 36, 48, 60 or 72 h).

[0021] In some embodiments, the method employs a recombinant microbial whole-cell catalytic system.

[0022] In some embodiments, the recombinant microbial whole-cell catalytic system comprises 5-30 mg / ml (e.g., 5, 10, 15, 20, 25, or 30 mg / mL) of recombinant microbial culture, 20-1000 mM (e.g., 20, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mM) of lactic acid, and 20-500 mM (e.g., 20, 70, 120, 170, 220, 270, 320, 370, 420, 470, or 500 mM) of formaldehyde.

[0023] In some embodiments, the recombinant microbial whole-cell catalytic system further includes a buffer system selected from any one of HEPES, Tris, MOPS, Tricine, or phosphate buffer, with a concentration of 5-50 mM (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM) and a pH of 5-10 (e.g., 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0).

[0024] In some embodiments, the reaction temperature of the recombinant microbial whole-cell catalytic system is 25-45°C (e.g., 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 or 45°C) and / or the reaction time is 1-72 h (e.g., 1, 2, 4, 8, 12, 24, 36, 48, 60 or 72 h).

[0025] In some embodiments, the recombinant microorganism comprises nucleic acid molecules encoding the lactate dehydrogenase, the aldolase, the decarboxylase, and the reductase.

[0026] In some embodiments, the recombinant microorganism is a microorganism with a missing or inactivated operon in the formaldehyde detoxification system. In this invention, the microorganism with a missing or inactivated operon in the formaldehyde detoxification system refers to a microorganism in which the core gene cluster frmRAB in the formaldehyde detoxification system has been knocked out. frmRAB consists of three cis-arranged genes: frmR, frmA, and frmB.

[0027] In some embodiments, the starting strain of the recombinant microorganism is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, lactic acid bacteria, and Pseudomonas putida.

[0028] In some embodiments, the recombinant microbial culture comprises the recombinant microorganism cultured under conditions suitable for the expression of the lactate dehydrogenase, aldolase, decarboxylase, and reductase.

[0029] In some embodiments, the method employs a recombinant microbial fermentation system.

[0030] In some embodiments, the recombinant microbial fermentation system includes recombinant microorganisms and a culture medium.

[0031] In some embodiments, the culture medium comprises a medium supplemented with 20-500 mM (e.g., 20, 70, 120, 170, 220, 270, 320, 370, 420, 470 or 500 mM) formaldehyde and 20-1000 mM (e.g., 20, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM) lactic acid.

[0032] In some embodiments, the culture medium comprises LB medium or M9 medium supplemented with 20-500 mM (e.g., 20, 70, 120, 170, 220, 270, 320, 370, 420, 470 or 500 mM) formaldehyde and 20-1000 mM (e.g., 20, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mM) lactic acid.

[0033] In some embodiments, the recombinant microorganism comprises nucleic acid molecules encoding the lactate dehydrogenase, the aldolase, the decarboxylase, and the reductase.

[0034] In some embodiments, the recombinant microorganism is a microorganism with a missing or inactivated operon in the formaldehyde detoxification system.

[0035] In some embodiments, the starting strain of the recombinant microorganism is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, lactic acid bacteria, and Pseudomonas putida.

[0036] In some embodiments, the lactic acid includes L-lactic acid, D-lactic acid, a mixture of L / D lactic acid, or a salt thereof (such as its sodium, potassium, or ammonium salt).

[0037] A second aspect of the present invention provides a recombinant microorganism or a culture thereof, the recombinant microorganism comprising nucleic acid molecules encoding lactate dehydrogenase, aldolase, decarboxylase and reductase, wherein the lactate dehydrogenase comprises lactate dehydrogenase classified under EC1.1.1.-, the aldolase comprises aldolase classified under EC 4.1.2.-, the reductase comprises reductase classified under EC 1.1.1.-, and the decarboxylase comprises decarboxylase classified under EC 4.1.1.-.

[0038] In some embodiments, the lactate dehydrogenase includes one or more of L-lactate dehydrogenase (EC: 1.1.1.27) and its variants, D-lactate dehydrogenase (EC: 1.1.1.28) and its variants.

[0039] In some embodiments, the lactate dehydrogenase includes one or more of L-lactate dehydrogenase or variants thereof from Lactobacillus pentosus, and L-lactate dehydrogenase or variants thereof from Mycoplasma synoviae.

[0040] In some embodiments, the L-lactate dehydrogenase from *Lactobacillus pentosus* comprises an amino acid sequence as shown in SEQ ID No: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it, and / or The L-lactate dehydrogenase from Mycoplasma synoviae comprises an amino acid sequence as shown in SEQ ID No: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0041] In some embodiments, the aldolase includes one or more of type I aldolases or variants thereof, type II aldolases or variants thereof.

[0042] In some embodiments, the aldolase comprises one or more of type II aldolases or variants thereof from the genus *Potassium*.

[0043] In some embodiments, the type II aldolase from the genus *Saprophyticus* comprises an amino acid sequence as shown in SEQ ID No: 3 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0044] In some embodiments, the decarboxylase includes one or more of keto acid decarboxylase (EC 4.1.1.1) or its variants.

[0045] In some embodiments, the decarboxylase includes one or more keto acid decarboxylases or variants thereof derived from Lactococcus lactis.

[0046] In some embodiments, the ketoacid decarboxylase variant of the lactococcus lactis comprises an amino acid sequence as shown in SEQ ID No: 4 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0047] In some embodiments, the reductase includes one or more of 1,3-propanediol dehydrogenase (EC 1.1.1.202) or variants thereof.

[0048] In some embodiments, the reductase comprises one or more of 1,3-propanediol dehydrogenase or variants thereof from Klebsiella pneumoniae.

[0049] In some embodiments, the 1,3-propanediol dehydrogenase from Klebsiella pneumoniae comprises an amino acid sequence as shown in SEQ ID No: 5 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with it.

[0050] In some embodiments, the recombinant microorganism is a microorganism with a missing or inactivated operon in the formaldehyde detoxification system.

[0051] In some embodiments, the starting strain of the recombinant microorganism is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, lactic acid bacteria, and Pseudomonas putida.

[0052] In some embodiments, the recombinant microbial culture comprises the recombinant microorganism cultured under conditions suitable for the expression of the lactate dehydrogenase, aldolase, decarboxylase, and reductase.

[0053] The third aspect of the present invention provides the use of the method of the first aspect or the recombinant microorganism or its culture of the second aspect in the preparation of 1,3-propanediol.

[0054] Compared with the prior art, the present invention has at least the following technical effects: 1) Eliminating the rate-limiting bottleneck: Directly using formaldehyde as a substrate avoids the inefficient rate-limiting step in the methanol-to-formaldehyde conversion, resulting in a simpler and more stable reaction process; 2) Strong system stability: The NAD self-balancing closed-loop design makes the reaction system less prone to imbalance and highly feasible for scale-up; 3) Inexpensive and sustainable raw materials: Lactic acid is widely available and low in cost, aligning with the concept of green manufacturing; 4) Green, low-carbon, and environmentally friendly: Only one molecule of carbon dioxide is released throughout the process, achieving near-zero waste generation at high conversion rates, significantly reducing product purification costs, and demonstrating excellent environmental friendliness and atom economy; 5) Outstanding industrialization potential: The simple pathway, controllable costs, and strong stability provide a new and feasible solution for the large-scale green manufacturing of 1,3-propanediol. Attached Figure Description

[0055] Figure 1 The biosynthetic pathway of 1,3-propanediol is shown.

[0056] Figure 2 The HPLC chromatogram shows the synthesis of 1,3-propanediol from formaldehyde and S-lactic acid using an in vitro multi-enzyme system. Detailed Implementation

[0057] This invention provides a biosynthetic method for 1,3-propanediol based on the self-equilibrium synthesis of NAD cofactor. Using lactic acid and formaldehyde as initial substrates, the synthesis is completed through a four-step enzymatic cascade reaction. The basic synthetic route is as follows: Figure 1 The process includes the following steps: 1) Lactic acid is dehydrogenated to pyruvate under the catalysis of lactate dehydrogenase, and NADH is generated simultaneously; 2) Pyruvate and formaldehyde undergo a condensation reaction under the catalysis of aldolase to generate 2-keto-4-hydroxybutyric acid; 3) 2-keto-4-hydroxybutyric acid is decarboxylated under the catalysis of decarboxylase to generate 3-hydroxypropanal; 4) 3-hydroxypropanal is reduced to 1,3-propanediol under the catalysis of reductase using the NADH generated in step 1, and NAD is regenerated. + 5) Achieving a dynamic balance of NAD cofactors throughout the process through a coupled closed loop of lactate dehydrogenation to produce NADH and terminal reduction to consume NADH. The biosynthesis method in this invention can be implemented through in vitro multi-enzyme catalysis, whole-cell catalysis by recombinant microorganisms, or recombinant microbial fermentation.

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0059] Definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0060] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0061] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0062] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0063] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0064] Unless otherwise specified, the experimental or measurement methods used in the embodiments of this invention shall be performed as follows: Determination of lactic acid and 1,3-propanediol: After the enzymatic reaction, the protein was denatured by heating at 95°C, the denatured enzyme was removed by centrifugation, and the sample was filtered through a 0.22 μm filter membrane and diluted with distilled water to a concentration below 50 mM. The final concentration was determined using an Aminex HPX-87H column (300). The column was analyzed by HPLC with a mobile phase of 5 mM H2SO4, a flow rate of 0.6 mL / min, a column temperature of 15 °C, and a RID differential detector at 35 °C.

[0065] The culture medium used in the embodiments of the present invention is as follows: (1) M9 Inorganic Salt Medium (also known as M9 medium): The basic components (1L) include: 47.8mM Na2HPO4, 22mM 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 H3BO3, 0.081μM MnCl2; 20g / L glucose and 2g / L yeast extract.

[0066] (2) LB medium (1L): 10g peptone, 10g sodium chloride, 5g yeast extract, pH 7.0.

[0067] The sequence information involved in the embodiments of the present invention is shown in Table 1: Table 1:

[0068] Example 1: Screening of key enzymes and plasmid construction in the biosynthetic pathway of 1,3-propanediol This invention introduces formaldehyde into the synthesis of 1,3-propanediol; the basic synthetic route is as follows: Figure 1 1,3-propanediol is synthesized from formaldehyde and S or R-lactic acid as substrates via dehydrogenase, aldolase, decarboxylase and reductase catalysis.

[0069] In this embodiment, the dehydrogenase is a dehydrogenase that catalyzes the dehydrogenation of lactate to pyruvate, specifically L-lactate dehydrogenase (Uniprot entry P56511) (SEQ ID No: 1) from Lactobacillus pentosus, named Lp Ldh, or L-lactate dehydrogenase (Uniprot entry Q4A5V1) from Mycoplasma synoviae (SEQ ID No: 2), is named Ms Ldh.

[0070] In this embodiment, the aldolase is an aldolase capable of catalyzing the reaction of pyruvate and formaldehyde to produce 2-keto-4-hydroxybutyrate, specifically a type II aldolase of the genus *Uniprot* (Uniprot entry A0A7D8V3B9, SEQ ID No:3), named... Vh Ald.

[0071] In this embodiment, the decarboxylase is an enzyme capable of catalyzing the decarboxylation reaction of 2-keto-4-hydroxybutyric acid to generate 3-hydroxypropionaldehyde. Specifically, it is a mutant (SEQ ID No:4) of keto acid decarboxylase (Uniprot entry Q6QBS4) from Lactococcus lactis. This mutant has a single-point mutation V461I relative to keto acid decarboxylase (Uniprot entry Q6QBS4). The mutant is named... Ll KdcA-V461I.

[0072] In this embodiment, the reductase is an enzyme that catalyzes the reduction of 3-hydroxypropanal to 1,3-propanediol, specifically the 1,3-propanediol dehydrogenase from Klebsiella pneumoniae (Uniprot entry Q59477, SEQ ID No: 5), named... Kp DhaT.

[0073] (1) Plasmid construction and preparation of expression strains Will Lp Ldh、 Ms Ldh、 Vh Ald, Ll KdcA-V461I, Kp The DhaT encoding gene was synthesized by Genewiz using NdeI and XhoI restriction sites and constructed into the pET16b vector. Codon optimization was performed using the E. coli system, and the resulting recombinant strain was transformed into the BL21(DE3) expression strain (Qingke Biotechnology, catalog number TSC-E01) to obtain the recombinant strain. Lp Ldh-BL21(DE3) Ms Ldh-BL21(DE3) Vh Ald-BL21(DE3) Ll KdcA-V461I-BL21(DE3) and Kp DhaT-BL21(DE3).

[0074] (2) Bacterial culture and induced expression 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.

[0075] Expanded culture and expression: Transfer 4 mL of bacterial culture to 800 mL of LB medium containing resistant bacteria (100 μg / mL ampicillin), incubate at 37°C and 220 rpm for 3-4 h until OD500. 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 after centrifugation at 6000 rpm for 15 minutes.

[0076] (3) Protein purification The target protein (i.e., the key enzyme in the synthetic pathway) prepared in this embodiment was... Lp Ldh、 Ms Ldh、 Vh Ald, Ll KdcA-V461I and Kp DhaT protein, tagged with histidine, was purified using a two-step process involving nickel ion chelate affinity chromatography coupled with size exclusion desalting chromatography. The entire purification process was performed using a protein purification system (Suzhou Insys Intelligent Technology Co., Ltd., model: Unique Autopure15s-L1). The specific purification steps are as follows.

[0077] (3.1) Cell lysis treatment The bacterial precipitate was fully resuspended in Lysis Buffer at a mass-to-volume ratio of 1 g:10 mL. The Lysis Buffer formulation consisted of 50 mM potassium dihydrogen phosphate, 10 mM imidazole, 300 mM sodium chloride, and 10% glycerol (v / v), with the pH adjusted to 8.0. The resuspended bacterial solution was then subjected to ultrasonic lysis in an ice-water bath. The ultrasonic parameters were set as follows: power 260 W, ultrasonic duration 3 s, interval 7 s, and total ultrasonic treatment time 15-30 min. The ultrasonic duration could be adjusted flexibly according to the clarity of the lysed bacterial cells until the bacterial solution was clear and free of significant turbidity and precipitation, indicating complete bacterial cell disruption.

[0078] (3.2) Preparation of lysis supernatant After ultrasonic lysis of the bacterial cells, the lysis system was centrifuged at 4 ℃ and 10,000 rpm for 30 min to thoroughly remove bacterial fragments, undone cells, and insoluble impurities, and the clear crude protein supernatant was collected. The obtained supernatant was filtered through a 0.22 μm aqueous filter membrane (Merck Millipore, catalog number: SLGP033RB) to obtain a protein sample solution that can be directly loaded.

[0079] (3.3) Nickel ion chelate affinity chromatography purification Target protein-specific purification was performed using a 5 mL HiTrap HP nickel affinity chromatography column (Cytiva, catalog number: 17524802). The column was first equilibrated with 20 mL Lysis Buffer at a flow rate of 1.0 mL / min. The filtered protein sample solution was then loaded at a constant flow rate of 1.0 mL / min. The target protein was immobilized within the column by the specific binding of the His-tag to nickel ions. After loading, the column was continuously washed with Wash Buffer to remove non-specifically bound proteins. The Wash Buffer formulation was: 50 mM potassium dihydrogen phosphate, 75 mM imidazole, 300 mM sodium chloride, 10% glycerol (v / v), pH 8.0. Washing continued until no significant absorption signal was observed at 280 nm using a UV detector, indicating complete elution of contaminating proteins. The target protein was then eluted using Buffer B, which consisted of 50 mM potassium dihydrogen phosphate, 500 mM imidazole, 300 mM sodium chloride, 10% glycerol (v / v), and pH 8.0. The eluted fractions were collected in segments, and the purity of each fraction was determined by SDS-PAGE electrophoresis. Elution buffers containing high-purity target protein were then selected.

[0080] (3.4) Protein desalting and buffer replacement To remove high concentrations of imidazole impurities from the eluent, a 5 mL HiTrap Desalting column (Cytiva, catalog number: 17140801) was used to desalt and replace the buffer in the purified protein sample. The desalting column was first pretreated by rinsing with 15 mL of ultrapure water, then fully equilibrated with 20 mL of Storage Buffer. The Storage Buffer formulation was: 20 mM HEPES, 300 mM sodium chloride, 10% glycerol (v / v), pH 7.5. The protein sample was loaded onto the column, and eluted with Storage Buffer at a flow rate of 1.0 mL / min. A280 UV absorbance was monitored in real time to accurately identify and collect the protein elution peak components, obtaining the high-purity target protein after desalting and purification.

[0081] (3.5) Cofactor replenishment, incubation and protein preservation The collected target proteins were re-examined by SDS-PAGE electrophoresis to confirm their purity. Since protein purification can easily lead to the loss of some naturally binding cofactors, cofactors were supplemented according to protein type to ensure the biological activity of the target proteins: NAD+ for dehydrogenases; MgCl2 for aldolases; TPP for decarboxylases; and NADH for reductases. The molar ratio of cofactor to protein in each protein sample was 20:1. After cofactor addition, the protein system was incubated at 4 ℃ for 2-4 h, gently mixing during this time to ensure complete binding of cofactors to proteins. After incubation, the protein samples were aliquoted and sealed in a -80 ℃ ultra-low temperature freezer for storage.

[0082] Example 2: Synthesis of 1,3-propanediol using formaldehyde and S-lactic acid as substrates via an in vitro multi-enzyme system The key enzymes of the synthetic pathway, purified and aliquoted as described in Example 1, were used to synthesize 1,3-propanediol from formaldehyde and S-lactic acid using an in vitro multi-enzyme system. Because the key enzymes of the synthetic pathway prepared in Example 1 underwent cofactor supplementation, adding them to the in vitro multi-enzyme system simultaneously introduces a certain amount of cofactors into the system. For example, when the in vitro multi-enzyme system is supplemented with... Lp When Ldh is used, the ratio will be 20:1 (NAD). + : Lp The molar ratio of Ldh to NAD + .

[0083] 1. Enzyme cascade systems with added exogenous cofactors The reaction conditions for the synthesis of 1,3-propanediol using formaldehyde and S-lactic acid as substrates in an enzyme cascade system were as follows: 20 mM MEPES, pH 7.5, 200 mM S-lactic acid, 0.5 mM TPP, 5 mM MgCl2, and 0.5 mM NAD. + 2mg / ml Lp Ldh or 2mg / ml Ms Ldh, 1 mg / ml Vh Ald, 2mg / ml Ll KdcA-V461I and 2mg / ml Kp For DhaT, 50 mM formaldehyde was added to the reaction system initially, followed by 50 mM formaldehyde every 3 hours for a total of 4 additions. The reaction temperature was 37°C, and the total reaction time was 12 hours. The reaction mixture contained 0.5 mM TPP, 5 mM MgCl2, and 0.5 mM NAD. + All of them are added from external sources and do not include the cofactors introduced due to the cofactor completion in Example 1.

[0084] The yield of 1,3-propanediol was monitored by HPLC and found to be 0.86 g / L.Lp Ldh group) and 0.68 g / L ( Ms Ldh group), HPLC chromatogram is shown in Figure 2 .

[0085] 2. In vitro multi-enzyme system with all exogenous cofactors removed 1,3-Propanediol was synthesized using formaldehyde and S-lactic acid as substrates under the following reaction conditions: 20 mM HEPES, pH 7.5, 200 mM S-lactic acid, 3 mg / ml Lp Ldh, 1.5 mg / ml Vh Ald, 3mg / ml Ll KdcA-V461I and 3 mg / ml KpDhaT were reacted with 50 mM formaldehyde added to the reaction system at the beginning of the reaction, and 50 mM formaldehyde was added every 10 hours for a total of 4 additions of formaldehyde. The reaction temperature was 30℃ and the total reaction time was 40 hours.

[0086] The yield of 1,3-propanediol was monitored by HPLC and found to be 0.78 g / L.

[0087] Example 3: Synthesis of 1,3-propanediol by microbial fermentation or whole-cell catalysis Construction of functional plasmids and bacterial strains: The encoding genes of LpLdh and VhAld were synthesized by Genewiz using whole-genome synthesis and constructed into the multiple cloning sites MCS-1 and MCS-2 of the pRSFDuet-1 vector using double restriction sites of BamHI and HindIII and NdeI and XhoI, respectively, to obtain the plasmid pRSFDuet-1- Lp Ldh- Vh Ald.

[0088] The encoding genes of LlKdcA-V461I and KpDhaT were synthesized by Genewiz using whole-genome synthesis and constructed into the multiple cloning sites MCS-1 and MCS-2 of the pETDuet-1 vector using double restriction sites of BamHI and HindIII and NdeI and XhoI, respectively, to obtain the plasmid pETDuet-1- Ll KdcA-V461I- Kp DhaT.

[0089] Strain construction: The core gene cluster frmRAB in the formaldehyde detoxification system of BL21(DE3) strain (Qingke Biotechnology, catalog number TSC-E01) was knocked out. It consists of three cis-arranged genes: frmR, frmA, and frmB. The knockout method was based on the literature GreenChem., 2023, 25, 7662.

[0090] Construction of recombinant strains: BL21 (DE3)△frmA electroporation competent cells were prepared using the glycerol washing method. Recombinant plasmids pRSFDuet-1-LpLdh-VhAld and pETDuet-1-LlKdcA-V461I-KpDhaT were diluted to 500 ng / μL, mixed evenly, and 800-1000 ng of total plasmid was mixed with 100 μL of competent cells for co-electroporation. After electroporation, 350 μL of LB medium was added for recovery, and the mixture was incubated at 37℃ for 45 min at 200 rpm. The cells were then plated on LB agar plates containing ampicillin (100 μg / mL) and kanamycin (50 μg / mL) and incubated overnight at 37℃. Engineered strains carrying both recombinant plasmids were screened.

[0091] Recombinant strain culture and induced expression: 1. Recombinant strains were cultured and induced to express using LB medium. Inoculate the single clone into 4 mL of LB medium, add kanamycin (50 μg / mL) and ampicillin (100 μg / mL) according to the resistance of the transformed plasmid, and incubate overnight at 37°C and 220 rpm.

[0092] Expanded culture and expression: Transfer 4 mL of bacterial culture to 800 mL of LB medium containing antibiotic resistance (50 μg / mL kanamycin and 100 μg / mL ampicillin), 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 after centrifugation at 6000 rpm for 15 minutes.

[0093] The collected bacterial cells were used for the enzymatic reaction. The reaction system was as follows: 20 mM HEPES pH 7.5, 500 mM S-lactic acid, 20 mg / ml wet cells, initial formaldehyde concentration of 50 mM, then 50 mM formaldehyde was added every 6 h, with a final formaldehyde concentration of 500 mM, reaction temperature of 37℃, and total reaction time of 72 h.

[0094] After the reaction, centrifuge and collect the supernatant. Filter through a 0.22 μM filter membrane and dilute the sample to below 50 mM concentration with distilled water. Then, use an Aminex HPX-87H column (300...) HPLC analysis was performed using a 7.8 mm column (7.8 mM H₂SO₄ as the mobile phase, 0.6 mL / min flow rate, 15 °C column temperature, and a RID differential detector at 35 °C. The yield of 1,3-propanediol was determined to be 0.84 g / L.

[0095] 2. Recombinant strains were cultured and induced to express using M9 medium. Inoculate the single clone into 4 mL of M9 medium, add kanamycin (50 μg / mL) and ampicillin (100 μg / mL) according to the resistance of the transformed plasmid, and incubate overnight at 37°C and 220 rpm.

[0096] Expanded culture and expression: 4 mL of bacterial culture was transferred to 200 mL of M9 resistant medium (50 μg / mL kanamycin and 100 μg / mL ampicillin), and cultured at 37°C and 220 rpm until OD500 reached. 600 After reaching a concentration of 0.3, 0.2 mM IPTG was added, followed by cooling to 30°C and incubation for 6 hours. Then, 50 mM formaldehyde and 200 mM S-lactic acid were added. Formaldehyde was added again at 25 mM every 6 hours for a total of 8 additions. The reaction was terminated after 60 hours. After the reaction, the supernatant was collected by centrifugation, filtered through a 0.22 μM filter membrane, and diluted with distilled water to a concentration below 50 mM. The sample was then analyzed using an Aminex HPX-87H column (300). HPLC analysis was performed on a column (7.8 mm) with 5 mM H2SO4 as the mobile phase, a flow rate of 0.6 mL / min, a column temperature of 15 °C, and a RID differential detector at 35 °C.

[0097] HPLC analysis showed that the yield of 1,3-propanediol was 0.11 g / L.

[0098] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method comprising the following enzymatic catalytic steps: (i) Converting lactic acid to pyruvate; (ii) The generated pyruvate condenses with formaldehyde to form 2-keto-4-hydroxybutyric acid; (iii) The generated 2-keto-4-hydroxybutyric acid undergoes decarboxylation to generate 3-hydroxypropanal; (iv) The generated 3-hydroxypropanal is reduced to 1,3-propanediol.

2. The method according to claim 1, characterized in that, In step (i), the EC classification is used. 1.1.1.- Lactate dehydrogenase enables the conversion of lactate to pyruvate; Preferably, the lactate dehydrogenase includes one or more of L-lactate dehydrogenase (EC: 1.1.1.27) and its variants, and D-lactate dehydrogenase (EC: 1.1.1.28) and its variants; Preferably, the lactate dehydrogenase comprises one or more of L-lactate dehydrogenase or variants thereof from Lactobacillus pentosus, and L-lactate dehydrogenase or variants thereof from Mycoplasma synoviae. Preferably, the L-lactate dehydrogenase from *Lactobacillus pentosus* comprises an amino acid sequence as shown in SEQ ID No: 1 or an amino acid sequence having at least 85% sequence identity with it, and / or The L-lactate dehydrogenase from Mycoplasma synoviae comprises an amino acid sequence as shown in SEQ ID No: 2 or an amino acid sequence having at least 85% sequence identity with it. Preferably, in step (ii), the EC classification is used. The aldolase 4.1.2.- generates pyruvate which condenses with formaldehyde to form 2-keto-4-hydroxybutyric acid; Preferably, the aldolase includes one or more of type I aldolase or variants thereof, type II aldolase or variants thereof; Preferably, the aldolase comprises one or more of type II aldolases or variants thereof from the genus *Saprophytica*. Preferably, the type II aldolase from the genus *Pyrophyllaria* comprises an amino acid sequence as shown in SEQ ID No: 3 or an amino acid sequence having at least 85% sequence identity with it; Preferably, in step (iii), the EC classification is used. 4.1.1.- The decarboxylase produces 2-keto-4-hydroxybutyric acid, which is then decarboxylated to generate 3-hydroxypropionaldehyde; Preferably, the decarboxylase includes keto acid decarboxylase (EC). 4.1.1.1) or one or more of its variants; Preferably, the decarboxylase comprises one or more keto acid decarboxylases or variants thereof derived from Lactococcus lactis; Preferably, the keto acid decarboxylase variant of the lactococcus lactis comprises an amino acid sequence as shown in SEQ ID No: 4 or an amino acid sequence having at least 85% sequence identity with it; Preferably, in step (iv), the EC classification is used. The reductase 1.1.1.- reduces the generated 3-hydroxypropanal to 1,3-propanediol; Preferably, the reductase comprises 1,3-propanediol dehydrogenase (EC... 1.1.1.202) or one or more of its variants; Preferably, the reductase comprises one or more of Klebsiella pneumoniae 1,3-propanediol dehydrogenase or variants thereof; Preferably, the 1,3-propanediol dehydrogenase from Klebsiella pneumoniae comprises an amino acid sequence as shown in SEQ ID No: 5 or an amino acid sequence having at least 85% sequence identity with it.

3. The method according to claim 1, characterized in that, The method employs an in vitro multi-enzyme catalytic system; Preferably, the in vitro multi-enzyme catalytic system comprises: 0-10 mM divalent metal ions, 0-2 mM thiamine pyrophosphate, 0-2 mM NAD+, 1-3 mg / mL lactate dehydrogenase, 0.5-2 mg / mL aldolase, 0.5-3 mg / mL decarboxylase, 0.5-3 mg / mL reductase, 20-1000 mM lactate, and 20-500 mM formaldehyde; Preferably, the in vitro multi-enzyme catalytic system comprises: 2-5 mg / mL lactate dehydrogenase, 1-3 mg / mL aldolase, 2-5 mg / mL decarboxylase, 2-5 mg / mL reductase, 20-1000 mM lactate, and 20-500 mM formaldehyde; Preferably, the in vitro multi-enzyme catalytic system further includes a buffer system, wherein the buffer system is selected from any one of HEPES, Tris, MOPS, Tricine or phosphate buffer, with a concentration of 5-50 mM and a pH of 5-10; Preferably, the reaction temperature of the in vitro multi-enzyme catalytic system is 25-45℃ and / or the reaction time is 1-72h.

4. The method according to claim 1, characterized in that, The method employs a recombinant microbial whole-cell catalytic system; Preferably, the recombinant microbial whole-cell catalytic system comprises 5-30 mg / ml of recombinant microbial culture, 20-1000 mM lactic acid, and 20-500 mM formaldehyde; Preferably, the recombinant microbial whole-cell catalytic system further includes a buffer system, wherein the buffer system is selected from any one of HEPES, Tris, MOPS, Tricine or phosphate buffer, with a concentration of 5-50 mM and a pH of 5-10; Preferably, the reaction temperature of the recombinant microbial whole-cell catalytic system is 25-45℃ and / or the reaction time is 1-72h.

5. The method according to claim 1, characterized in that, The method employs a recombinant microbial fermentation system; Preferably, the recombinant microbial fermentation system includes recombinant microorganisms and a culture medium; Preferably, the culture medium includes a culture medium supplemented with 20-500 mM formaldehyde and 20-1000 mM lactic acid.

6. The method according to claim 4 or 5, characterized in that, The recombinant microorganism contains nucleic acid molecules encoding the lactate dehydrogenase, the aldolase, the decarboxylase, and the reductase; Preferably, the recombinant microorganism is a microorganism with a missing or inactivated operon in the formaldehyde detoxification system; Preferably, the starting strain of the recombinant microorganism is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, lactic acid bacteria, and Pseudomonas putida. Preferably, the recombinant microbial culture comprises the recombinant microorganism cultured under conditions suitable for the expression of the lactate dehydrogenase, aldolase, decarboxylase, and reductase.

7. The method according to any one of claims 1-6, characterized in that, The lactic acid includes L-lactic acid, D-lactic acid, a mixture of L / D lactic acid, or a salt thereof.

8. A recombinant microorganism or a culture thereof, said recombinant microorganism comprising nucleic acid molecules encoding lactate dehydrogenase, aldolase, decarboxylase and reductase as described in claim 2.

9. The recombinant microorganism or its culture according to claim 8, characterized in that, The recombinant microorganisms are microorganisms with missing or inactivated operons in the formaldehyde detoxification system; Preferably, the starting strain of the recombinant microorganism is selected from any one of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, lactic acid bacteria, and Pseudomonas putida. Preferably, the recombinant microbial culture comprises the recombinant microorganism cultured under conditions suitable for the expression of the lactate dehydrogenase, aldolase, decarboxylase, and reductase.

10. Use of the method of any one of claims 1-7 or the recombinant microorganism or its culture as described in claim 8 or 9 in the preparation of 1,3-propanediol.

Images