阅读说明：本技术 一种从甲酸到甲醛和/或甲醇的生物合成方法 (Biosynthesis method from formic acid to formaldehyde and/or methanol ) 是由 马延和 蔡韬 孙红兵 吕娟博 王钦宏 于 2020-08-24 设计创作，主要内容包括：本发明公开了一种从甲酸合成甲醛和/或甲醇的新途径,通过不同化学反应的组合,实现了甲酸到甲醛和/或甲醇的高效合成。本发明的新途径需要的能量更少(每生产1摩尔甲醛需要消耗1摩尔ATP到ADP),且效率更高,1小时即可生产2mM甲醛或4.83mM甲醇。本发明的新途径可通过与已有途径整合,实现利用二氧化碳、生物质、天然气等原料合成复杂化合物和液体燃料的目的。(The invention discloses a new way for synthesizing formaldehyde and/or methanol from formic acid, which realizes the high-efficiency synthesis of the formic acid to the formaldehyde and/or the methanol by the combination of different chemical reactions. The new route of the invention requires less energy (1 mole ATP to ADP is consumed per 1 mole formaldehyde produced) and is more efficient, producing 2mM formaldehyde or 4.83mM methanol in 1 hour. The new way of the invention can be integrated with the existing way, and the purpose of synthesizing complex compounds and liquid fuels by utilizing raw materials such as carbon dioxide, biomass, natural gas and the like is realized.)

1. A method for the biosynthesis of formaldehyde from formic acid comprising the steps of:

step (1): formic acid or its salt is used as raw material, and is converted into formyl phosphate under the catalysis of enzyme;

step (2): converting formyl phosphate obtained in the step (1) into formyl coenzyme A under the catalysis of enzyme; and

and (3): converting the formyl coenzyme A obtained in the step (2) into formaldehyde under the catalysis of enzyme;

preferably, the enzyme used in step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formyl phosphate; the enzyme used in the step (2) is an enzyme with the function of catalyzing formyl phosphate to be converted into formyl coenzyme A; the enzyme used in the step (3) is an enzyme having a function of catalyzing the conversion of formyl coenzyme A into formaldehyde.

2. The process according to claim 1, wherein the formate salt is an alkali or alkaline earth metal salt of formic acid, such as sodium formate, potassium formate, lithium formate, magnesium formate or calcium formate.

3. A method according to claim 1 or 2, wherein steps (1) to (3) are carried out in steps, or wherein any two or three adjacent steps are carried out simultaneously.

4. The method according to claim 3, wherein the steps (1) to (3) are carried out simultaneously, for example, the reaction system comprises a substrate of formic acid or formate, an enzyme having a function of catalyzing the conversion of formic acid to formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate to formyl CoA, and an enzyme having a function of catalyzing the conversion of formyl CoA to formaldehyde.

5. The method according to any one of claims 1 to 4, wherein the reaction system of step (3) or the reaction systems in which steps (1) to (3) are carried out simultaneously optionally comprise a supplementary enzyme for assisting NADH regeneration, such as Formate Dehydrogenase (FDH).

6. A method for the biosynthesis of methanol from formate, comprising the steps of:

step (1): formic acid or its salt is used as raw material, and is converted into formyl phosphate under the catalysis of enzyme;

step (2): converting formyl phosphate obtained in the step (1) into formyl coenzyme A under the catalysis of enzyme;

and (3): converting the formyl coenzyme A obtained in the step (2) into formaldehyde under the catalysis of enzyme; and

and (4): converting the formaldehyde obtained in the step (3) into methanol under the catalysis of enzyme;

preferably, the enzyme used in step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formyl phosphate; the enzyme used in the step (2) is an enzyme with the function of catalyzing formyl phosphate to be converted into formyl coenzyme A; the enzyme used in the step (3) is an enzyme with the function of catalyzing the conversion of formyl coenzyme A into formaldehyde; the enzyme used in the step (4) is an enzyme having a function of catalyzing the conversion of formaldehyde into methanol.

7. A process according to claim 6, wherein the formate salt is an alkali or alkaline earth metal salt of formic acid, such as sodium formate, potassium formate, lithium formate, magnesium formate or calcium formate.

8. A method according to claim 6 or 7, wherein steps (1) to (4) are carried out in steps, or wherein any two, three or four adjacent steps are carried out simultaneously.

9. The method according to claim 8, wherein the steps (1) to (4) are carried out simultaneously, for example, the reaction system comprises a substrate of formic acid or formate, an enzyme having a function of catalyzing the conversion of formic acid to formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate to formyl CoA, an enzyme having a function of catalyzing the conversion of formyl CoA to formaldehyde and an enzyme having a function of catalyzing the conversion of formaldehyde to methanol.

10. The method according to any one of claims 6 to 9, wherein the reaction system of step (3) or step (4) or the reaction system in which steps (1) to (4) are carried out simultaneously optionally comprises a helper enzyme for assisting the regeneration of NADH, such as Formate Dehydrogenase (FDH).

Technical Field

The invention belongs to the technical field of biosynthesis, and particularly relates to a biosynthesis method for producing formaldehyde and/or methanol from formic acid.

Background

Formic acid, formaldehyde, methanol are products of carbon dioxide reduction to varying degrees, which are important carbon feedstocks that can be used to synthesize a variety of compounds. The chemical activity of formaldehyde and methanol is higher than that of formic acid, and the subsequent conversion and application are facilitated.

The methods reported so far are mainly to convert formic acid into formaldehyde or methanol by formaldehyde dehydrogenase or methanol dehydrogenase in the natural pathway. However, this method is disadvantageous in that free energy is unfavorable and a large amount of reducing power is required to obtain a small amount of accumulation of formaldehyde or alcohol.

In addition to the natural pathway, the artificial pathway consisting of acetyl-coa synthetase and acetaldehyde dehydrogenase effects the reduction of formate to formaldehyde, but requires the consumption of more energy (1 mole ATP to AMP is consumed for every 1 mole of formaldehyde produced).

In addition, the artificial pathway using acetate kinase and aspartate semialdehyde dehydrogenase consumes less energy (1 mole ATP to ADP is consumed per 1 mole of formaldehyde produced), but the formaldehyde yield is low, only 0.0163 mM.

Thus, there is a need in the art for a less energy intensive, high yield biosynthetic process from formic acid to formaldehyde and/or methanol.

Disclosure of Invention

In order to solve the problems in the prior art, in one aspect, the present invention provides a method for biosynthesis of formic acid to formaldehyde, comprising the steps of:

step (1): formic acid or its salt is used as raw material, and is converted into formyl phosphate under the catalysis of enzyme;

step (2): converting formyl phosphate obtained in the step (1) into formyl coenzyme A under the catalysis of enzyme; and

and (3): converting the formyl coenzyme A obtained in the step (2) into formaldehyde under the catalysis of enzyme.

According to an embodiment of the present invention, the enzyme used in step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formyl phosphate.

According to an embodiment of the invention, the formate salt is an alkali metal or alkaline earth metal salt of formic acid, such as sodium formate, potassium formate, lithium formate, magnesium formate, calcium formate, and the like.

According to an embodiment of the present invention, the enzyme used in step (2) is an enzyme having a function of catalyzing the conversion of formyl phosphate into formyl-CoA.

According to an embodiment of the present invention, the enzyme used in step (3) is an enzyme having a function of catalyzing the conversion of formyl-CoA into formaldehyde.

According to an embodiment of the present invention, steps (1) to (3) may be performed in steps, or any two or three adjacent steps may be performed simultaneously.

According to an embodiment of the present invention, steps (1) to (3) are performed simultaneously, for example, the reaction system comprises a substrate of formic acid or formate, an enzyme having a function of catalyzing the conversion of formic acid to formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate to formyl CoA, and an enzyme having a function of catalyzing the conversion of formyl CoA to formaldehyde.

According to an embodiment of the invention, the reaction system further comprises NaCl, Mg²⁺、Zn²⁺ATP, NADH, CoA, mercaptoethanol, etc.

According to an embodiment of the invention, the reaction system has a pH of 6.5 to 8.5, such as 7 to 8, said pH environment being provided, for example, with Hepes buffer.

According to an embodiment of the present invention, the reaction system in step (3) or the reaction systems in which steps (1) to (3) are carried out simultaneously may optionally further comprise a helper enzyme for assisting NADH regeneration, such as Formate Dehydrogenase (FDH).

In another aspect, the present invention also provides a method for biosynthesis of formic acid to methanol, comprising the steps of:

step (1): formic acid or its salt is used as raw material, and is converted into formyl phosphate under the catalysis of enzyme;

step (2): converting formyl phosphate obtained in the step (1) into formyl coenzyme A under the catalysis of enzyme;

and (3): converting the formyl coenzyme A obtained in the step (2) into formaldehyde under the catalysis of enzyme; and

and (4): converting the formaldehyde obtained in the step (3) into methanol under the catalysis of enzyme.

According to an embodiment of the present invention, the enzyme used in step (1) is an enzyme having a function of catalyzing the conversion of formic acid into formyl phosphate.

According to an embodiment of the invention, the formate salt is an alkali metal or alkaline earth metal salt of formic acid, such as sodium formate, potassium formate, lithium formate, magnesium formate, calcium formate, and the like.

According to an embodiment of the present invention, the enzyme used in step (2) is an enzyme having a function of catalyzing the conversion of formyl phosphate into formyl-CoA.

According to an embodiment of the present invention, the enzyme used in step (3) is an enzyme having a function of catalyzing the conversion of formyl-CoA into formaldehyde.

According to an embodiment of the present invention, the enzyme used in step (4) is an enzyme having a function of catalyzing the conversion of formaldehyde into methanol.

According to an embodiment of the present invention, steps (1) to (4) may be performed in steps, or any adjacent two, three or four steps may be performed simultaneously.

According to an embodiment of the present invention, the steps (1) to (4) are performed simultaneously, and the reaction system comprises a substrate of formic acid or formate, an enzyme having a function of catalyzing the conversion of formic acid into formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate into formyl coenzyme A, an enzyme having a function of catalyzing the conversion of formyl coenzyme A into formaldehyde, and an enzyme having a function of catalyzing the conversion of formaldehyde into methanol.

According to an embodiment of the invention, the reaction system further comprises NaCl, Mg²⁺、Zn²⁺ATP, NADH, CoA, mercaptoethanol, etc.

According to an embodiment of the invention, the reaction system has a pH of 6.5 to 8.5, such as 7 to 8, said pH environment being provided, for example, with Hepes buffer.

According to an embodiment of the present invention, the reaction system in step (3) or step (4) or the reaction system in which steps (1) to (4) are carried out simultaneously may further optionally comprise a supplementary enzyme for assisting NADH regeneration, such as Formate Dehydrogenase (FDH).

In the context of the present invention, when the expression "enzyme having a function of catalyzing the conversion of a substance a into a substance B" is used, it means that the enzyme can catalyze the reaction of converting a substance a into a substance B, which may be a one-step reaction or a multi-step reaction, and the enzyme may be an enzyme required for any one step of the reaction of producing substance B from substance a, and thus, the enzyme may be a single enzyme catalyzing the one-step reaction or a combination of enzymes catalyzing one or more steps of the multi-step reaction. The amino acid sequence and source of the enzyme having the catalytic function are not particularly limited as long as they can fulfill the catalytic function. Specifically, "an enzyme having a function of catalyzing the conversion of formate to formyl phosphate" refers to an enzyme that can catalyze the phosphorylation of formate, including but not limited to acetate kinase (ACKA, EC 2.7.2.1) and formate kinase (EC 2.7.2.6), which can be derived from, but not limited to, Escherichia coli, Salmonella, Clostridium, Methanosarcina, and other different species. "enzyme having a function of catalyzing the conversion of formyl phosphate to formyl CoA" refers to an enzyme that can catalyze the reaction of converting formyl phosphate to formyl CoA, including but not limited to, phosphate acetyltransferase (PTA, EC 2.3.1.8), which can be derived from, but not limited to, Escherichia coli, Clostridium, Thermotoga, Methanosarcina, and the like, among various species. "enzyme having a function of catalyzing the conversion of formyl-coa to formaldehyde" refers to an enzyme that can catalyze the conversion of formyl-coa to formaldehyde, including but not limited to acetaldehyde dehydrogenase (ACDH, EC 1.2.1.10), which can be derived from, but not limited to, listeria, pseudomonas, acinetobacter, giardia, and other different species. "enzyme having a function of catalyzing the conversion of formaldehyde into methanol" refers to an enzyme that can catalyze a reaction in which formaldehyde is reduced into methanol, and includes, but is not limited to, alcohol dehydrogenase (ADH, EC 1.1.1.1; EC 1.1.1; EC 1.1.2; EC 1.1.1.71; EC 1.1.2.8), methanol dehydrogenase (EC 1.1.1.244; EC 1.1.2.7; EC 1.1.2.B2), L-threonine-3-dehydrogenase (EC 1.1.1.103), cyclohexanol dehydrogenase (EC 1.1.1.245), and n-butanol dehydrogenase (EC 1.1.2.9), which may be derived from, but not limited to, different species such as Pichia pastoris, Candida, Streptomyces, Corynebacterium glutamicum, Escherichia coli, Rhodococcus, and Tourella.

The enzymes used in the context of the present invention may be in the form of crude enzyme solutions, crude enzyme solution lyophilized powders, pure enzymes or whole cells. The crude enzyme solution, the crude enzyme solution freeze-dried powder, the pure enzyme or the whole cells can be obtained commercially or prepared according to a method known in the literature or a conventional method in the field; for example, the crude enzyme solution freeze-dried powder and the pure enzyme are prepared according to the method comprising the following steps: expressing the enzyme in a host cell to obtain a recombinant cell; cracking the recombinant cells to obtain the crude enzyme solution, the crude enzyme solution freeze-dried powder or pure enzyme; the whole cells are prepared according to a method comprising the following steps: and expressing the enzyme in a host cell to obtain a recombinant cell, namely the whole cell.

In the context of the present invention, the term "step-wise" means that after the preceding reaction step is completed, the product is optionally purified, and then an enzyme or a desired component that catalyzes the subsequent reaction step is added to perform the subsequent reaction step; the term "simultaneously carrying out" means that an enzyme catalyzing each reaction involved is fed into a reactor together with a substrate to carry out the reaction at the start of the reaction. The "adjacent" step means that the product of a preceding step can be used as a reactant for a subsequent step, and the two steps can be referred to as "adjacent".

Advantageous effects

The new way for synthesizing formaldehyde and/or methanol by formic acid or the salt thereof realizes the high-efficiency synthesis of the formic acid or the salt thereof to the formaldehyde and/or the methanol by the combination of different chemical reactions. The new route of the invention requires less energy (1 mole ATP to ADP is consumed per 1 mole formaldehyde produced) and is more efficient, producing 2mM formaldehyde or 4.83mM methanol in 1 hour. The new way of the invention can be integrated with the existing way, and the purpose of synthesizing complex compounds and liquid fuels by utilizing raw materials such as carbon dioxide, biomass, natural gas and the like is realized.

Drawings

FIG. 1 shows a comparison of formaldehyde production by the novel pathway of the present invention for the synthesis of formaldehyde from formate with the natural pathway and the acetyl-CoA synthetase pathway.

Figure 2 shows the methanol yield of the new route to methanol from formic acid according to the invention.

Detailed Description

The process for the synthesis of formaldehyde and methanol from formic acid according to the invention will be described in further detail with reference to the following examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

Example 1 Synthesis of Formaldehyde from formic acid

The new route for the synthesis of formaldehyde from formic acid is shown below:

first, a catalyst (i.e., an enzyme) that catalyzes each chemical reaction in the pathway is selected (see table 1), but is not limited to the catalysts listed in table 1. Then, different catalysts are combined to establish a corresponding reaction system, and after a period of reaction, the yield of formaldehyde is detected.

Information on the enzymes used in example 1 is listed in table 1, and acetate kinase, phosphate acetyltransferase, acetaldehyde dehydrogenase, formate dehydrogenase are obtained by means of PCR or gene synthesis and cloned into pET20b, pET21b and pET28a vectors (Novagen, Madison, WI) by the method of Simple Cloning (You, c., et al (2012), "Simple Cloning via Direct Transformation of PCR products (DNAMultimer) to Escherichia coli and Bacillus subtilis," appl.environ.Microbiol.78(5): 1593-1595), respectively, to obtain the corresponding expression vectors acdt 28a-ACKA, pET28a-PTA, pET21 pe 21 b-FDH, t20 b-h. All four plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

TABLE 1 catalysts for the chemical reaction of the novel formic acid to formaldehyde route

The formaldehyde yield was measured as follows: after adding 50. mu.L of an appropriately diluted solution to be tested to 200. mu.L of water, adding 25. mu.L of an acetylacetone solution (100mL of an acetylacetone solution containing 0.5mL of acetylacetone, 50g of ammonium acetate and 6mL of glacial acetic acid) and reacting at 60 ℃ for 15min, 200. mu.L of the supernatant was centrifuged to determine the OD414 value, and the amount of formaldehyde generated by the new route was calculated from the standard formaldehyde curve.

In addition, the natural pathway and the already disclosed capacity of the artificial pathway for converting formic acid to formaldehyde by acetyl-CoA synthetase were also tested as comparative examples 1-1 and 1-2, respectively.

Novel routes 1-2-3 of example 1: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M,50mM sodium formate, 10mM ATP, 0.5mM NADH, 0.1mM CoA, 2mM mercaptoethanol, 0.24mg/mL ACKA, 1.2mg/mL PTA, 0.2mg/mL ACDH, 0.024mg/mL FDH (formate dehydrogenase, assisted NADH regeneration). The reaction was carried out for 1 hour, and the yield of formaldehyde was 2 mM.

The natural route of comparative example 1-1: the reaction system is a Hepes buffer solution with pH7.5 of 100mM, NaCl of 100mM and Mg²⁺ 5mM，Zn²⁺10 μ M, FADH 4mg/mL, NADH 100mM, sodium formate 250 mM. The reaction was carried out for 3 hours, and the yield of formaldehyde was 0.1 mM.

Acetyl-coa synthetase pathway of comparative examples 1-2: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺ 5mM，Zn²⁺ 10μM，ATP 2mM，NAD⁺1.5mM, CoA 0.1mM, ACS 3.7mg/mL, ACDH 0.2mg/mL, FDH 0.024mg/mL (formate dehydrogenase, helper enzyme, for regenerating NADH), PPase 0.1mg/mL (inorganic pyrophosphatase, helper enzyme), sodium formate 50 mM. The reaction was carried out for 1 hour, and the yield of formaldehyde was 0.6 mM.

Comparison of the formaldehyde yields for the new route of example 1 with the synthetic routes of comparative examples 1-1 and comparative examples 1-2 is shown in figure 1. As can be seen from FIG. 1, the novel approach of the present invention for the synthesis of formaldehyde from formic acid results in significantly higher formaldehyde yields in a shorter time than the natural approach and the acetyl-CoA synthetase approach.

Example 2 novel route to methanol from formic acid

The new route to methanol from formic acid is shown below:

first, a catalyst that catalyzes each chemical reaction in the pathway is selected (see table 2), but is not limited to the catalysts listed in table 2. Then, different catalysts are combined to establish a corresponding reaction system, and after a period of reaction, the yield of the methanol is detected.

The information on the enzymes used in example 2 is given in Table 2, and the alcohol dehydrogenases were purchased from Sigma (https:// www.sigmaaldrich.com/china-mainland. html). The other enzymes were constructed and expressed in the same manner as in example 1.

TABLE 2 catalysts for the chemical reaction of the new formic acid to methanol route

The methanol yield was measured as follows: the amount of methanol produced in the new route was calculated from a standard curve using an Agilent 7890A chromatograph (containing a FID detector), nitrogen as the carrier gas, 18kPa (0.4mL/min) as the pressure, HP-FFAP (25 m.times.0.32 mm. times.0.5 μm) as the column, 75 ℃ for 10min as the initial column temperature, 150 ℃ and 300 ℃ for the injector and detector, respectively, and 1 μ L/needle as the sample size.

The reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺ 5mM，Zn²⁺10 μ M,100mM sodium formate, 10mM ATP, 0.5mM NADH, 0.1mM CoA, 2mM mercaptoethanol, 0.24mg/mL ACKA, 1.2mg/mL PTA, 0.2mg/mL ACDH, 0.024mg/mL FDH (formate dehydrogenase, assisted NADH regeneration), 0.15kU/mL LADH; the reaction time was 1 hour, and the yield of methanol was 4.83 mM. The yield of methanol is shown in FIG. 2.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.