A multi-enzyme cascade method for catalyzing synthesis of isobutanol by C1 and application thereof

Abstract

The present application relates to a kind of catalytic C1 synthesis isobutanol multi-enzyme cascade method and its application, the method includes: (1) with formaldehyde or methanol as raw material, by aldehyde condensation enzyme condensation into dihydroxy acetone;(2) dihydroxy acetone is converted into pyruvic acid by the artificial metabolic pathway constructed;(3) pyruvic acid is converted into isobutanol by the artificial metabolic pathway constructed.The method of the present application is coupled by C1→dihydroxy acetone, dihydroxy acetone→pyruvic acid, and pyruvic acid→isobutanol module cascade, by each reaction module condition optimization and with system integration, realizes the efficient conversion of C1 compound to isobutanol, provides a new path for constructing the green biological manufacturing system of isobutanol with C1 raw material full-chain synthesis.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to a multi-enzyme cascade method for catalyzing the synthesis of isobutanol from C1 cells and its application. Background Technology

[0002] The unsustainability of fossil fuels and the related energy crisis are becoming increasingly prominent, driving the development of biofuels as a key strategic direction for ensuring energy security, reducing greenhouse gas emissions, and addressing global climate change. Efforts are being made to vigorously promote renewable energy substitution and actively expand the application of advanced biofuels and other clean energy sources in the transportation sector.

[0003] Currently, the mainstream biofuel products on the market mainly include bioethanol. However, these fuels generally suffer from low energy density and strong hydrophilicity (e.g., ethanol is miscible with water), which limits their wider application. In contrast, bioisobutanol (C4H) is a more suitable option. 10 Isobutanol (O) possesses higher volumetric energy density and lower hygroscopicity, demonstrating significant advantages as a next-generation fossil fuel alternative. Its branched structure better matches the molecular characteristics of existing petroleum-based fuels, thus exhibiting promising prospects for industrial application. Furthermore, as an important platform compound, isobutanol has been widely used in the synthesis of pharmaceutical intermediates, dyes, and other products in the fine chemical industry; in energy conversion, it can serve as a key precursor to isobutylene, a major component of liquefied petroleum gas and a fundamental raw material for petrochemicals.

[0004] Currently, biosynthesis of isobutanol utilizes natural isobutanol synthesis pathways based on the valine metabolic pathway or the Calvin cycle, as well as artificial isobutanol precursor synthesis pathways based on carbohydrate substrates. However, it still faces the following technical bottlenecks: ① The complexity of natural metabolic networks makes it difficult to precisely control carbon flow distribution; ② The use of glycosyl raw materials easily triggers "food-energy" controversies, limiting its industrialization. Therefore, constructing an artificial isobutanol pathway using C1 compounds (formaldehyde or methanol) as renewable raw materials will avoid the controversies faced by artificial pathways based on carbohydrate substrates, while also aligning with sustainable development strategies. Furthermore, constructing an artificial isobutanol pathway using C1 as a raw material also requires consideration of enzyme catalytic module design, directional carbon flow control, and energy matching supply to improve the overall carbon conversion rate and isobutanol synthesis yield. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a multi-enzyme cascade method for catalytic synthesis of isobutanol from C1 compounds and its application. Addressing the challenges of limited metabolic pathways for isobutanol synthesis from C1 feedstocks and difficulties in controlling the equilibrium of multi-enzyme reactions in existing technologies, this invention provides a multi-enzyme cascade reaction pathway for catalytic synthesis of isobutanol from C1 compounds. This pathway consists of a cascade coupling of modules C1 → dihydroxyacetone, dihydroxyacetone → pyruvate, and pyruvate → isobutanol. Through optimization of the conditions of each reaction module and system integration, highly efficient conversion of C1 compounds to isobutanol is achieved, providing a novel pathway for constructing a green biomanufacturing system for the full-chain synthesis of isobutanol from C1 feedstocks.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multi-enzyme cascade method for catalytic synthesis of isobutanol from C1 cells, the method comprising: (1) Using formaldehyde or methanol as raw materials, dihydroxyacetone is condensed by aldolase. (2) Dihydroxyacetone is converted into pyruvate through a constructed artificial metabolic pathway; (3) Pyruvate is converted into isobutanol through a constructed artificial metabolic pathway.

[0007] In this invention, C1 (formaldehyde or methanol) is used as a raw material. First, formaldehyde is condensed into dihydroxyacetone by aldolase (C1 → dihydroxyacetone). Then, dihydroxyacetone is converted into pyruvate by a cascade catalysis of dihydroxyacetone kinase and the like (dihydroxyacetone → pyruvate). Finally, pyruvate is synthesized into isobutanol by a cascade catalysis of acetolactate synthase and the like through condensation and carboxylation reactions (pyruvate → isobutanol). Methanol is used as a C1 raw material in a multi-enzyme cascade pathway, and it can be converted into formaldehyde by methanol oxidase.

[0008] Preferably, in step (1), when the raw material is formaldehyde, it is condensed into dihydroxyacetone using an aldolase. Preferably, in step (1), when the raw material is methanol, methanol oxidase is used to convert it into formaldehyde, and then aldolase is used to condense it into dihydroxyacetone.

[0009] Preferably, the aldolase comprises aldolase PfFLS.

[0010] In one specific embodiment of the present invention, the aldolase used is PfFLS, which is derived from *Pseudomonas fluorescens*. Pseudomonas fluorescens In this invention, any enzymes capable of catalyzing the condensation of formaldehyde into dihydroxyacetone can be used in the above-mentioned pathway.

[0011] Preferably, the methanol oxidase includes methanol oxidase GtAOX.

[0012] In one specific embodiment of the present invention, the methanol oxidase used is GtAOX, derived from *Cladosporium filamentosa*. Gloeophyllum trabeum In this invention, any enzymes capable of catalyzing the oxidation of methanol to formaldehyde can be used in the above-mentioned pathway.

[0013] Preferably, in step (2), the artificial metabolic pathway includes the conversion of dihydroxyacetone to dihydroacetone phosphate under the catalysis of dihydroxyacetone kinase, the conversion of dihydroacetone phosphate to pyruvate under the catalysis of methylglyoxal synthase, and the conversion of pyruvate to pyruvate under the catalysis of aldehyde dehydrogenase.

[0014] Preferably, the dihydroxyacetone kinase includes dihydroxyacetone kinase PpDHAK.

[0015] In one specific embodiment of the present invention, the dihydroxyacetone kinase used is PpDHAK, derived from Pichia pastoris. Pichia pastoris In this invention, any enzymes capable of catalyzing the conversion of dihydroxyacetone to dihydroxyacetone phosphate can be used in the above-mentioned pathway.

[0016] Preferably, the methylglyoxal synthase comprises methylglyoxal synthase CbMGSA.

[0017] In one specific embodiment of the present invention, the methylglyoxal synthase used is CbMGSA, derived from Coxsella burgdorferi. Coxiella burnetiid In this invention, any enzymes capable of catalyzing the conversion of dihydroxyacetone phosphate to acetone aldehyde can be used in the above pathway.

[0018] Preferably, the aldehyde dehydrogenase includes aldehyde dehydrogenase ApAlDH.

[0019] In one specific embodiment of the present invention, the aldehyde dehydrogenase used is ApAlDH, which is derived from *Acetobacter pasteurella*. Acetobacter pasteurianus In this invention, any enzymes capable of catalyzing the conversion of pyruvaldehyde to pyruvate can be used in the above-mentioned pathway.

[0020] Preferably, in step (3), the artificial metabolic pathway includes the conversion of pyruvate to acetolactate under the catalysis of acetolactate synthase, the conversion of acetolactate to 2,3-dihydroxyisovalerate under the catalysis of ketool acid reductase, and the conversion of 2,3-dihydroxyisovalerate to dihydroxy acid dehydratase under the catalysis of dihydroxy acid dehydratase. - Ketopentyl isocyanate - Ketoamyl isocyanate is converted to isobutyraldehyde under the catalysis of ketoamyl isocyanate decarboxylase, and isobutyraldehyde is converted to isobutanol under the catalysis of aldehyde reductase.

[0021] Preferably, the acetolactate synthase includes acetolactate synthase BsALS.

[0022] In one specific embodiment of the present invention, the acetolactate synthase used is BsALS, derived from Bacillus subtilis. Bacillus subtilis In this invention, all enzymes that catalyze the synthesis of acetolactate from pyruvate can be used in the above-mentioned pathway.

[0023] Preferably, the ketoalkanoate reductase includes ketoalkanoate reductase MrKARI.

[0024] In one specific embodiment of the present invention, the keto-alcoholic acid reductase used is MrKARI, derived from *Thermus rubrum*. Meiothermus ruber In this invention, enzymes that catalyze the isomerization of acetolactate to 2,3-dihydroxyisovalerate can all be used in the above pathway.

[0025] Preferably, the dihydroxy acid dehydratase includes dihydroxy acid dehydratase SmDHAD.

[0026] In one specific embodiment of the present invention, the dihydroxy acid dehydratase used is SmDHAD, which is derived from Streptococcus mutans. Streptococcus mutans In this invention, there is a catalytic method for the dehydration of 2,3-dihydroxyisovalerate to produce... Enzymes related to the function of ketopentyl isocyanate can all be used in the above pathways.

[0027] Preferably, the ketopentyl isocyanate decarboxylase includes ketopentyl isocyanate decarboxylase LlKDC.

[0028] In one specific embodiment of the present invention, the ketopentidoic acid decarboxylase used is LlKDC, which is derived from Lactococcus lactis. Lactococcus lactis In this invention, all enzymes that catalyze the decarboxylation of ketopentyl isocyanate to generate isobutyraldehyde can be used in the above-mentioned pathway.

[0029] Preferably, the aldehyde reductase includes aldehyde reductase L1ADH.

[0030] In one specific embodiment of the present invention, the aldehyde reductase used is L1ADH, which is derived from Lactococcus lactis. Lactococcus lactis In this invention, enzymes that catalyze the reduction of isobutyraldehyde to isobutanol can be used in the above-mentioned pathway.

[0031] In a second aspect, the present invention provides a method for synthesizing isobutanol by a multi-enzyme cascade catalysis of C1, the method comprising synthesizing isobutanol using the multi-enzyme cascade method for synthesizing isobutanol by catalysis of C1 as described in the first aspect.

[0032] Preferably, the method includes: mixing raw materials, thiamine pyrophosphate, MgCl2, ATP, and NAD. + Flavin adenine dinucleotide, NADH, and a complex enzyme are mixed and reacted in a buffer solution to produce isobutanol.

[0033] Preferably, the complex enzyme includes PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH.

[0034] Preferably, the mass ratio of PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH is 10:(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-30):(0.1-30):(0.1-10):(0.1-10). The (0.1-30) can be, for example, 0.1, 1, 5, 10, 15, 20, 25, or 30. The (0.1-10) can be, for example, 0.1, 1, 2, 4, 6, 8, or 10.

[0035] Preferably, when the raw material is methanol, the complex enzyme includes GtAOX, PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH.

[0036] Preferably, the mass ratio of GtAOX, PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH is 1:(0.1-30):(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-30):(0.1-30):(0.1-10):(0.1-10). The (0.1-30) can be, for example, 0.1, 1, 5, 10, 15, 20, 25, or 30. The (0.1-10) can be, for example, 0.1, 1, 2, 4, 6, 8, or 10.

[0037] Preferably, the buffer solution comprises any one or a combination of at least two of the following: HEPES buffer, citrate buffer, PB buffer, Tris-HCl buffer, isobutanolamine buffer, diglycine buffer, or sodium carbonate buffer.

[0038] Preferably, the concentration of the buffer solution is 2-100 mM. The 2-100 mM can be, for example, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM.

[0039] Preferably, the pH value of the buffer solution is 4-9. The 4-9 can be, for example, 4, 5, 6, 7, 8, or 9.

[0040] Preferably, the concentration of thiamine pyrophosphate is 0.1-10.0 mM. For example, it can be 0.1 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM or 10 mM, etc.

[0041] Preferably, the concentration of MgCl2 is 0.1-100 mM. For example, it can be 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.

[0042] Preferably, the concentration of ATP is 0.1-100 mM. For example, it can be 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.

[0043] Preferably, the NAD + The concentration ranges from 0.1 to 100 mM. For example, it can be 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, etc.

[0044] Preferably, the concentration of the flavin adenine dinucleotide is 0.001-1.0 mM. For example, it can be 0.001 mM, 0.01 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM or 1.0 mM, etc.

[0045] Preferably, the concentration of NADH is 0.1-100 mM. For example, it can be 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.

[0046] Preferably, the concentration of the complex enzyme is 1-10 mg / mL. For example, it can be 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, or 10 mg / mL, etc.

[0047] Preferably, the concentration of the raw material is 0.1-100 mM. For example, it can be 0.1 mM, 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, etc.

[0048] Preferably, the reaction time is 1-24 h and the temperature is 25°C. o C-35 o C. The 1-24 h can be, for example, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, or 24 h, etc. The 25 o C-35 o C, for example, could be 25 o C, 27 o C, 29 o C, 31 o C, 33 o C or 35 o C, etc.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects: This invention differs from existing biosynthetic pathways based on valine metabolism and dependent on carbohydrate compounds. The multi-enzyme cascade pathway constructed in this invention uses C1 compounds as raw materials, achieves C–C bond construction through highly efficient aldolases, and further couples artificially designed cascade enzyme catalytic modules to convert C1 into isobutanol. This provides a brand-new technical path for the high-value utilization of C1 raw materials and fundamentally avoids the "food-energy" controversy, which has significant scientific significance and industrialization potential. Attached Figure Description

[0050] Figure 1 This is a diagram of the multi-enzyme cascade reaction pathway for the synthesis of isobutanol from C1.

[0051] Figure 2 The image shows the liquid phase detection results of the methanol-dihydroxyacetone module catalyzing the synthesis of dihydroxyacetone from methanol.

[0052] Figure 3 The image shows the liquid phase detection results of the formaldehyde → dihydroxyacetone module catalyzing the synthesis of dihydroxyacetone from formaldehyde.

[0053] Figure 4 This is a flowchart illustrating the process of synthesizing pyruvate from dihydroxyacetone using the dihydroxyacetone → pyruvate module.

[0054] Figure 5 This is a flowchart illustrating the process of synthesizing isobutanol from pyruvate using the pyruvate → isobutanol module. Detailed Implementation

[0055] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0056] Example 1 This embodiment establishes a multi-enzyme cascade catalytic method for the synthesis of isobutanol from C1 cells. The multi-enzyme cascade method for catalytic C1 synthesis of isobutanol constructed in this invention includes: using C1 (formaldehyde or methanol) as raw material, condensing formaldehyde or formaldehyde synthesized from methanol via methanol oxidase to dihydroxyacetone using aldolase; then converting dihydroxyacetone to pyruvate through a cascade catalysis of dihydroxyacetone kinase, methylglyoxal synthase, and aldehyde dehydrogenase; and finally synthesizing isobutanol from pyruvate through a cascade catalysis of acetolactate synthase, ketool acid reductase, dihydroxy acid dehydratase, ketoamyl isocarboxylase, and aldehyde reductase via condensation and carboxylation reactions. The specific process is as follows: Figure 1 As shown.

[0057] Example 2 This embodiment verifies the above-mentioned approach. (1) Synthesis of dihydroxyacetone When the raw material is methanol: At 30 °C, 30.0 mM substrate methanol and 200 mL of HEPES buffer (100 mM, pH 7.0) were added to a total volume of 1 mL. The reaction system contained 1 mg of methanol oxidase (GtAOX), 1 mg of aldolase (PfFLS), 1.0 mM thiamine pyrophosphate (TPP), and 10.0 mM MgCl2. The control group did not contain methanol oxidase (GtAOX). After 2 hours of reaction, the reaction was terminated with 0.5 M concentrated sulfuric acid. The amount of dihydroxyacetone produced was analyzed using a Shimadzu HPLC LC20A liquid chromatograph with a RID differential detector. Figure 2 As shown, the peak position of dihydroxyacetone is 27.7 min, and the concentration is 5.86 mM.

[0058] When the raw material is formaldehyde: At 30 °C, 30.0 mM formaldehyde, 1 mg aldolase PfFLS, 1.0 mM thiamine pyrophosphate (TPP), and 10.0 mM MgCl2 were added to 1 mL of HEPES buffer (100 mM, pH 7.0). The control group did not contain PfFLS. After 2 h of reaction, the reaction was quenched with 0.5 M concentrated sulfuric acid. The amount of dihydroxyacetone produced was analyzed using a Shimadzu HPLC LC20A with a RID differential detector. Figure 3As shown, the peak position of dihydroxyacetone is 27.7 min, and the concentration is 7.74 mM.

[0059] (2) Synthesis of pyruvate At 30 °C, 10.0 mM of substrate dihydroxyacetone and 50 mg of [unspecified substance] were added to a total volume of 1 mL of HEPES buffer (100 mM, pH 7.0). g dihydroxyacetone kinase PpDHAK, 50 g-methylglyoxal synthase CbMGSA, 50 g aldehyde dehydrogenase ApAlDH, 10.0 mM MgCl2, 20.0 mM coenzyme NAD + 10.0 mM ATP and 0.01 mM flavin adenine dinucleotide (FAD) were used. Samples were taken at different reaction times, and the reaction was quenched using 0.5 M concentrated sulfuric acid. The amount of pyruvate produced was analyzed using a Shimadzu HPLC LC20A liquid chromatograph with a RID differential detector. Figure 4 As shown, the peak position of pyruvate was 19.5 min, and the concentration was 4.30 mM.

[0060] (3) Synthesis of isobutanol At 30 °C, 10.0 mM substrate pyruvate and 50 mg of other additives were added to a total volume of 1 mL HEPES buffer (100 mM, pH 7.0). g acetyllactate synthase BsALS, 500 g dihydroxy acid dehydratase SmDHAD, 500 g ketool acid reductase MrKARI, 50 g-ketovalerate decarboxylase LlKDC, 50 The reaction mixture consisted of 10.0 mM aldehyde reductase L1ADH, 10.0 mM MgCl2, 20.0 mM coenzyme NADH, and 1.0 mM TPP. After 2 h, the reaction was terminated with 0.5 M concentrated sulfuric acid. The amount of isobutanol produced was analyzed using a Shimadzu Nexis GC-2030 gas chromatograph with a BID detector. Figure 5 As shown, the peak position of isobutanol is 12.4 min, and the concentration of the product is 4.68 mM.

[0061] Example 3 This embodiment demonstrates the synthesis of isobutanol from formaldehyde via a multi-enzyme cascade reaction. At 30 °C, 30.0 mM of substrate formaldehyde, 1 mg of aldolase PfFLS, and 50 mg of other components were added to a total volume of 1 mL of HEPES buffer (100 mM, pH 7.0). g dihydroxyacetone kinase PpDHAK, 50 g-methylglyoxal synthase CbMGSA, 50 g aldehyde dehydrogenase ApAlDH, 50 g acetyllactate synthase BsALS, 500 g dihydroxy acid dehydratase SmDHAD, 500 g ketool acid reductase MrKARI, 50 g-ketovalerate decarboxylase LlKDC, 50 g-aldol reductase LlADH, 20.0 mM coenzyme NAD + The reaction mixture consisted of 20.0 mM coenzyme NADH, 10.0 mM ATP, 1.0 mM thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), and 10.0 mM MgCl2. After 2 h of reaction, the reaction was terminated with 0.5 M concentrated sulfuric acid. The amount of isobutanol produced was analyzed using a Shimadzu Nexis GC-2030 gas chromatograph with a BID detector. The peak position of isobutanol was 12.4 min, and the concentration was 0.92 mM.

[0062] Example 4 This embodiment describes the synthesis of isobutanol from methanol or formaldehyde using a multi-enzyme cascade reaction as a catalytic feedstock. When the raw material is methanol: At 30 °C, 30.0 mM substrate methanol and 200 mL of HEPES buffer (100 mM, pH 7.0) were added to a total volume of 1 mL. g methanol oxidase GtAOX, 1 mg aldolase PfFLS, 50 g dihydroxyacetone kinase PpDHAK, 50 g-methylglyoxal synthase CbMGSA, 50 g aldehyde dehydrogenase ApAlDH, 50 g acetyllactate synthase BsALS, 500 g dihydroxy acid dehydratase SmDHAD, 500 g ketool acid reductase MrKARI, 50 g-ketovalerate decarboxylase LlKDC, 50 g aldehyde reductase LlADH (complex enzyme dosage of 2.5 mg / mL), 20.0 mM coenzyme NAD + The reaction mixture consisted of 20.0 mM coenzyme NADH, 10.0 mM ATP, 1.0 mM thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), and 10.0 mM MgCl2. After 8 h of reaction, the reaction was terminated with 0.5 M concentrated sulfuric acid, resulting in an isobutanol concentration of 1.2 mM.

[0063] When the raw material is formaldehyde: At 30°C, 30.0 mM of substrate formaldehyde, 1 mg of aldolase PfFLS, and 50 mg of other components were added to a total volume of 1 mL of HEPES buffer (100 mM, pH 7.0). g dihydroxyacetone kinase PpDHAK, 50 g-methylglyoxal synthase CbMGSA, 50 g aldehyde dehydrogenase ApAlDH, 50 g acetyllactate synthase BsALS, 500 g dihydroxy acid dehydratase SmDHAD, 500 g ketool acid reductase MrKARI, 50 g-ketovalerate decarboxylase LlKDC, 50 g aldehyde reductase LlADH (complex enzyme dosage of 2.3 mg / mL), 20.0 mM coenzyme NAD + The reaction was carried out with 20.0 mM coenzyme NADH, 10.0 mM ATP, 1.0 mM thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), and 10.0 mM MgCl2. After 8 h of reaction, the reaction was terminated with 0.5 M concentrated sulfuric acid, and the concentration of isobutanol produced increased to 2.6 mM.

[0064] Example 5 This embodiment demonstrates the synthesis of isobutanol from methanol of varying concentrations via a multi-enzyme cascade reaction. At 30 °C, different concentrations of methanol (30.0 mM, 45 mM, 60 mM, 100 mM) and 200 mM were added to a total volume of 1 mL HEPES buffer (100 mM, pH 7.0). g methanol oxidase GtAOX, 1 mg aldolase PfFLS, 50 g dihydroxyacetone kinase PpDHAK, 50 g-methylglyoxal synthase CbMGSA, 50 g aldehyde dehydrogenase ApAlDH, 50 g acetyllactate synthase BsALS, 500 g dihydroxy acid dehydratase SmDHAD, 500 g ketool acid reductase MrKARI, 50 g-ketovalerate decarboxylase LlKDC, 50 g aldehyde reductase LlADH (complex enzyme dosage 2.5 mg / mL), 20.0 mM coenzyme NAD +The reaction was carried out with 20.0 mM coenzyme NADH, 10.0 mM ATP, 1.0 mM thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), and 10.0 mM MgCl2 for 8 h. The reaction was then terminated with 0.5 M concentrated sulfuric acid, and the isobutanol concentrations were 1.2 mM, 2.1 mM, 3.2 mM, and 0.9 mM, respectively.

[0065] In summary, the pathway of this invention is formed by the cascade coupling of C1 → dihydroxyacetone, dihydroxyacetone → pyruvate, and pyruvate → isobutanol modules. Through optimization of the conditions of each reaction module and system integration, the efficient conversion of C1 compounds to isobutanol is achieved, providing a new path for constructing a green biomanufacturing system for the full-chain synthesis of isobutanol from C1 raw materials.

[0066] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A multi-enzyme cascade method for catalyzing the synthesis of isobutanol from C1, characterized in that, The method includes: (1) Using formaldehyde or methanol as raw materials, dihydroxyacetone is condensed by aldolase. (2) Dihydroxyacetone is converted into pyruvate through a constructed artificial metabolic pathway; (3) Pyruvate is converted into isobutanol through a constructed artificial metabolic pathway.

2. The multi-enzyme cascade method for catalytic C1 synthesis of isobutanol according to claim 1, characterized in that, In step (1), when the raw material is formaldehyde, it is condensed into dihydroxyacetone using an aldolase. Preferably, in step (1), when the raw material is methanol, methanol oxidase is used to convert it into formaldehyde, and then aldolase is used to condense it into dihydroxyacetone. Preferably, the aldolase includes aldolase PfFLS; Preferably, the methanol oxidase includes methanol oxidase GtAOX.

3. The multi-enzyme cascade method for catalytic C1 synthesis of isobutanol according to claim 1 or 2, characterized in that, In step (2), the artificial metabolic pathway includes the conversion of dihydroxyacetone to dihydroacetone phosphate under the catalysis of dihydroxyacetone kinase, the conversion of dihydroacetone phosphate to pyruvate under the catalysis of methylglyoxal synthase, and the conversion of pyruvate to pyruvate under the catalysis of aldehyde dehydrogenase.

4. The multi-enzyme cascade method for catalytic C1 synthesis of isobutanol according to claim 3, characterized in that, The dihydroxyacetone kinase includes dihydroxyacetone kinase PpDHAK; Preferably, the methylglyoxal synthase comprises methylglyoxal synthase CbMGSA; Preferably, the aldehyde dehydrogenase includes aldehyde dehydrogenase ApAlDH.

5. The multi-enzyme cascade method for catalytic C1 synthesis of isobutanol according to any one of claims 1-4, characterized in that, In step (3), the artificial metabolic pathway includes the conversion of pyruvate to acetolactate under the catalysis of acetolactate synthase, the conversion of acetolactate to 2,3-dihydroxyisovalerate under the catalysis of ketool acid reductase, and the conversion of 2,3-dihydroxyisovalerate to dihydroxy acid dehydratase under the catalysis of dihydroxy acid dehydratase. - Ketopentyl isocyanate - Ketoamyl isocyanate is converted to isobutyraldehyde under the catalysis of ketoamyl isocyanate decarboxylase, and isobutyraldehyde is converted to isobutanol under the catalysis of aldehyde reductase.

6. The multi-enzyme cascade method for catalytic C1 synthesis of isobutanol according to claim 5, characterized in that, The acetolactate synthase includes acetolactate synthase BsALS; Preferably, the ketoalkanoate reductase includes ketoalkanoate reductase MrKARI; Preferably, the dihydroxy acid dehydratase includes dihydroxy acid dehydratase SmDHAD; Preferably, the ketopentose decarboxylase includes ketopentose decarboxylase LlKDC; Preferably, the aldehyde reductase includes aldehyde reductase L1ADH.

7. A method for synthesizing isobutanol by C1 synthesis catalyzed by a multi-enzyme cascade, characterized in that, The method comprises synthesizing isobutanol using a multi-enzyme cascade method for catalytic C1 synthesis of isobutanol as described in any one of claims 1-6.

8. The method for synthesizing isobutanol by multi-enzyme cascade catalysis of C1 cells according to claim 7, characterized in that, The method includes: mixing raw materials, thiamine pyrophosphate, MgCl2, ATP, and NAD. + Flavin adenine dinucleotide, NADH, and a complex enzyme are mixed and reacted in a buffer solution to produce isobutanol.

9. The method for synthesizing isobutanol by multi-enzyme cascade catalysis of C1 cells according to claim 8, characterized in that, When the raw material is formaldehyde, the complex enzyme includes PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH; Preferably, the mass ratio of PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH is 10:(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-30):(0.1-30):(0.1-10):(0.1-10). Preferably, when the raw material is methanol, the complex enzyme includes GtAOX, PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC, and LlADH; Preferably, the mass ratio of GtAOX, PfFLS, PpDHAK, CbMGSA, ApAlDH, BsALS, MrKARI, SmDHAD, LlKDC and LlADH is 1: (0.1-30):(0.1-10):(0.1-10):(0.1-10):(0.1-10):(0.1-30):(0.1-30):(0.1-10):(0.1-10).

10. The method for synthesizing isobutanol by a multi-enzyme cascade catalysis of C1 cells according to claim 8 or 9, characterized in that, The buffer solution includes any one or a combination of at least two of the following: HEPES buffer, citrate buffer, PB buffer, Tris-HCl buffer, isobutanolamine buffer, diglycine buffer, or sodium carbonate buffer. Preferably, the concentration of the buffer solution is 2-100 mM; Preferably, the pH value of the buffer solution is 4-9; Preferably, the concentration of thiamine pyrophosphate is 0.1-10.0 mM; Preferably, the concentration of MgCl2 is 0.1-100 mM; Preferably, the concentration of ATP is 0.1-100 mM; Preferably, the NAD + The concentration ranges from 0.1 to 100 mM; Preferably, the concentration of the flavin adenine dinucleotide is 0.001-1.0 mM; Preferably, the concentration of NADH is 0.1-100 mM; Preferably, the concentration of the complex enzyme is 1-10 mg / mL; Preferably, the concentration of the raw material is 0.1~100 mM; Preferably, the reaction time is 1-24 h and the temperature is 25-35 °C. o C.

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