Starch biosynthesis method

文档序号：355322 发布日期：2021-12-07 浏览：19次中文

阅读说明：本技术 一种淀粉的生物合成方法 (Starch biosynthesis method ) 是由马延和蔡韬孙红兵乔婧张璠张洁王钦宏于 2020-08-24 设计创作，主要内容包括：本发明提供了淀粉的生物合成方法,该方法可以实现从二羟丙酮、甲醛、甲酸以及甲醇等简单化合物至淀粉的全人工生物合成,通过与二氧化碳化学还原等方法偶联,甚至可以以二氧化碳作为起始原料实现淀粉的全人工生物合成。天然淀粉合成需要经过卡尔文循环,共需要21-22步反应,而本发明的方法只需要9-12步反应,减少近一半的反应步骤,可大大缩短生产周期。此外,本发明的方法可以利用高浓度、高密度的二氧化碳和高能量密度的电能和氢能,更适合工业生产模式,生产周期可从农业种植的数个月缩减到数天。(The invention provides a starch biosynthesis method, which can realize total artificial biosynthesis from simple compounds such as dihydroxyacetone, formaldehyde, formic acid, methanol and the like to starch, can be coupled with methods such as carbon dioxide chemical reduction and the like, and can even realize the total artificial biosynthesis of the starch by taking the carbon dioxide as a starting material. The synthesis of the natural starch needs Karlvin cycle, and 21-22 steps of reaction are needed, while the method only needs 9-12 steps of reaction, so that nearly half of reaction steps are reduced, and the production period can be greatly shortened. In addition, the method can utilize high-concentration and high-density carbon dioxide and high-energy-density electric energy and hydrogen energy, is more suitable for industrial production modes, and can shorten the production period from several months to several days of agricultural planting.)

1. A method for synthesizing starch (I), which comprises the steps of converting a compound D, namely dihydroxyacetone, into starch under the catalysis of a plurality of enzymes;

the steps include the steps of:

step (1): the compound D, namely dihydroxyacetone, is used as a raw material and is converted into a compound F, namely D-3-glyceraldehyde phosphate under the catalysis of one or more enzymes;

step (2): converting the compound F obtained in the step (1) into a compound I, namely D-glucose-6-phosphate under the catalysis of one or more enzymes; and

and (3): converting the compound I obtained in the step (2) into starch under the catalysis of one or more enzymes;

preferably, the enzyme used in step (1) is an enzyme or a combination of enzymes that catalyzes the conversion of dihydroxyacetone to D-3-glyceraldehyde phosphate via one or more reactions; for example, a combination of an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate to dihydroxyacetone phosphate and an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate to D-3-glyceraldehyde phosphate;

the enzyme used in the step (2) is an enzyme or a combination of enzymes which catalyze the conversion of D-3-glyceraldehyde phosphate into D-glucose-6-phosphate through one-step or multi-step reaction; for example, the following enzyme combinations are possible: enzyme combination (I-2-a): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate into D-fructose-1, 6-diphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate into D-fructose-6-phosphate, and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate; or a combination of enzymes (I-2-b): a combination of an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate;

the enzyme used in the step (3) is an enzyme or a combination of enzymes which catalyze the conversion of D-glucose-6-phosphate into amylose or amylopectin through one-step or multi-step reaction; for example, the following enzyme combinations are possible: enzyme combination (I-3-a): a combination of an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate and an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose; or a combination of enzymes (I-3-b): a combination of an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into adenosine diphosphate- α -D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose into amylose; optionally, the combination (I-3-a) or the combination (I-3-b) may further comprise an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

2. The method (I) according to claim 1, wherein step (1) comprises the following sub-steps:

step (1-1): compound D is converted to compound E, dihydroxyacetone phosphate, catalyzed by one or more enzymes (this reaction is denoted as reaction 9); and

step (1-2): converting the compound E obtained in the step (1-1) into a compound F, namely D-3-glyceraldehyde phosphate under the catalysis of one or more enzymes (the reaction is recorded as a reaction 10);

preferably, the enzyme used in step (1-1) is an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate; the enzyme used in the step (1-2) is an enzyme having a function of catalyzing dihydroxyacetone phosphate to be converted into D-3-glyceraldehyde phosphate;

the step (2) comprises the following sub-steps:

step (2-1): compound F is converted to compound H, i.e. D-fructose-6-phosphate, under the catalysis of one or more enzymes; and

step (2-2): converting the compound H obtained in the step (2-1) into a compound I, namely D-glucose-6-phosphate (the reaction is marked as reaction 15) under the catalysis of one or more enzymes;

preferably, the enzyme used in step (2-1) is an enzyme or a combination of enzymes that catalyzes the conversion of glyceraldehyde-D-3-phosphate to D-fructose-6-phosphate via one or more reactions; for example, it may be an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate to D-fructose-6-phosphate alone, or it may be an enzyme combination (I-2-1): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate into D-fructose-1, 6-diphosphate and an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate into D-fructose-6-phosphate; the enzyme used in the step (2-2) is an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate;

more preferably, step (2-1) may be performed as follows: compound F is converted into compound H under the catalysis of an enzyme having the function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate (the reaction is marked as reaction 13 or 14); alternatively, step (2-1) may be performed as follows: firstly, a compound F is converted into a compound G, namely D-fructose-1, 6-diphosphate under the catalysis of an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-diphosphate (the reaction is recorded as a reaction 11), and then the obtained compound G is converted into D-fructose-6-phosphate under the catalysis of an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate into D-fructose-6-phosphate (the reaction is recorded as a reaction 12);

the step (3) comprises the following sub-steps:

step (3-1): compound I is converted to compound J, i.e. α -D-glucose-1-phosphate, under the catalysis of one or more enzymes (this reaction is denoted as reaction 16);

step (3-2): converting the compound J obtained in the step (3-1) into a compound 1, namely amylose, under the catalysis of one or more enzymes;

and optionally step (3-3): converting the compound 1 obtained in the step (3-2) into a compound 2, namely amylopectin under the catalysis of one or more enzymes (the reaction is denoted as a reaction 20);

preferably, the enzyme used in step (3-1) is an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate;

the enzyme used in the step (3-2) is an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose; for example, it may be an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose alone, or it may be an enzyme combination (I-3-2): a combination of an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate to adenosine diphosphate- α -D-glucose and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose to amylose;

the enzyme used in the step (3-3) is an enzyme having a function of catalyzing the conversion of amylose into amylopectin;

more preferably, step (3-2) may be performed as follows: compound J, which is converted to compound 1, i.e. amylose, under the catalysis of an enzyme having the function of catalyzing the conversion of α -D-glucose-1-phosphate to amylose (this reaction is denoted as reaction 19); alternatively, step (3-2) may be performed as follows: firstly, compound J is converted to compound K, i.e., adenosine diphosphate- α -D-glucose, under the catalysis of an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate to adenosine diphosphate- α -D-glucose (this reaction is denoted as reaction 17); then, the obtained compound K is converted into amylose by the catalysis of an enzyme having a function of catalyzing the conversion of adenosine diphosphate-alpha-D-glucose into amylose (this reaction is referred to as reaction 18).

3. The process (I) according to claim 1 or 2, wherein the process (I) further comprises, before step (1), a step (0) of converting formaldehyde into compound D, dihydroxyacetone, catalyzed by one or more enzymes, which reaction is designated as reaction 8; more preferably, the enzyme used in step (0) is an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone.

4. The process (I) according to claim 3, wherein the process (I) further comprises, before step (0), a step (a) of converting methanol or formic acid, starting from methanol or formic acid, into formaldehyde, catalysed by one or more enzymes; more preferably, in the step (a), when methanol is used as a raw material to synthesize formaldehyde, the enzyme used is an enzyme having a function of catalyzing the conversion of methanol into formaldehyde; when formic acid is used as a raw material to synthesize formaldehyde, the enzyme used is an enzyme having a function of catalyzing the conversion of formic acid into formaldehyde, and may be, for example, an enzyme having a function of catalyzing the conversion of formic acid into formaldehyde alone or may be an enzyme combination (I-a-1): an enzyme having a function of catalyzing the conversion of formate to formyl-CoA with an enzyme having a function of catalyzing the conversion of formyl-CoA to formaldehyde, or a combination of enzymes (I-a-2): a combination of an enzyme having a function of catalyzing the conversion of formate into formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate into formyl coenzyme A, and an enzyme having a function of catalyzing the conversion of formyl coenzyme A into formaldehyde;

more preferably, when formaldehyde is synthesized from formic acid as a raw material in step (a), it may be performed according to any one of the following steps (a1), (a2) or (a 3):

step (a 1): formic acid is used as a raw material and is converted into formaldehyde under the catalysis of enzyme with the function of catalyzing the conversion of the formic acid into the formaldehyde (the reaction is marked as reaction 3);

step (a 2): firstly, formic acid is taken as a raw material and is converted into formyl coenzyme A under the catalysis of enzyme with the function of catalyzing the conversion of the formic acid into the formyl coenzyme A (the reaction is marked as reaction 4); then, formyl-coa is converted to formaldehyde under the catalysis of an enzyme having a function of catalyzing the conversion of formyl-coa to formaldehyde (this reaction is denoted as reaction 7); or

Step (a 3): firstly, formic acid is used as a raw material and is converted into formyl phosphate under the catalysis of enzyme with the function of catalyzing the conversion of the formic acid into the formyl phosphate (the reaction is marked as reaction 5); subsequently, formyl phosphate is converted to formyl-coa catalyzed by an enzyme having a function of catalyzing the conversion of formyl phosphate to formyl-coa (this reaction is denoted as reaction 6); then, formyl-coa is converted to formaldehyde by the catalysis of an enzyme having a function of catalyzing the conversion of formyl-coa to formaldehyde (this reaction is denoted as reaction 7).

5. A process (II) for the synthesis of starch comprising the steps of:

step 1): methanol is used as a raw material and is converted into amylose under the catalysis of a plurality of enzymes; and optionally step 2): amylose is used as a raw material and is converted into amylopectin under the catalysis of one or more enzymes;

preferably, the enzyme used in step 1) is a combination of enzymes that catalyze methanol synthesis of starch via multi-step reactions; for example, the following enzyme combinations are possible:

enzyme combination (II-1-a): an enzyme having a function of catalyzing conversion of methanol into formaldehyde, an enzyme having a function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, an enzyme having a function of catalyzing conversion of alpha-D-glucose-1-phosphate into adenosine diphosphate-alpha-D-glucose, and an enzyme having a function of catalyzing conversion of adenosine diphosphate-alpha-D- A combination of enzymes functional for the conversion of glucose to amylose;

enzyme combination (II-1-b): an enzyme having a function of catalyzing the conversion of methanol into formaldehyde, an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into alpha-fructose-6-phosphate, and a method for producing a process for producing a, A combination of an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate to adenosine diphosphate- α -D-glucose and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose to amylose;

enzyme combination (II-1-c): an enzyme having a function of catalyzing the conversion of methanol into formaldehyde, an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, a combination of an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate and an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose; or

Enzyme combination (II-1-d): an enzyme having a function of catalyzing conversion of methanol into formaldehyde, an enzyme having a function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, an enzyme having a function of catalyzing conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, and an enzyme having a function of catalyzing conversion of alpha-D-glucose-1-phosphate into alpha-D-glucose-1-phosphate A combination of enzymes functional for conversion of acid to amylose;

the enzyme used in the step 2) is an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

6. A process (III) for the synthesis of starch comprising the steps of:

step 1): methanol is used as a raw material and is converted into a compound D, namely dihydroxyacetone under the catalysis of one or more enzymes;

step 2): converting dihydroxyacetone obtained in the step 1) into amylose under the catalysis of one or more enzymes; and

optional step 3): converting the amylose obtained in the step 2) into amylopectin under the catalysis of one or more enzymes;

preferably, the enzyme used in step 1) is the enzyme combination (III-1): a combination of an enzyme having a function of catalyzing the conversion of methanol to formaldehyde and an enzyme having a function of catalyzing the conversion of formaldehyde to dihydroxyacetone;

the enzymes used in step 2) are the following enzyme combinations:

enzyme combination (III-2-a): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate, a combination of an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into adenosine diphosphate- α -D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose into amylose;

enzyme combination (III-2-b): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into adenosine diphosphate-alpha-D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate into adenosine diphosphate A combination of enzymes that convert-alpha-D-glucose diphosphate to amylose;

enzyme combination (III-2-c): a combination of an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into glyceraldehyde D-3-phosphate, an enzyme having a function of catalyzing the conversion of glyceraldehyde D-3-phosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, and an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose; or

Enzyme combination (III-2-d): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, a combination of an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, and an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose;

the enzyme used in the step 3) is an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

7. A process (IV) for the synthesis of starch comprising the steps of:

the method comprises the following steps: formic acid is used as a raw material and is converted into dihydroxyacetone under the catalysis of one or more enzymes;

step two: converting dihydroxyacetone obtained in the step I into amylose under the catalysis of one or more enzymes; and

optional step (c): converting the amylose obtained in the step two into amylopectin under the catalysis of one or more enzymes;

preferably, the enzyme used in step (r) is an enzyme or a combination of enzymes that catalyses the conversion of formate to dihydroxyacetone in one or more reactions, and may for example be the following enzyme combinations:

enzyme combination (IV-1-a): a combination of an enzyme having a function of catalyzing the conversion of formic acid to formaldehyde and an enzyme having a function of catalyzing the conversion of formaldehyde to dihydroxyacetone;

enzyme combination (IV-1-b): a combination of an enzyme having a function of catalyzing the conversion of formic acid 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 dihydroxyacetone; or

Enzyme combination (IV-1-c): a combination of an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, and an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate;

the enzyme used in step (II) is an enzyme or a combination of enzymes that catalyze the conversion of dihydroxyacetone into amylose through one or more reactions, and may be, for example, the following enzyme combinations:

enzyme combination (IV-2-a): a combination of an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into glyceraldehyde D-3-phosphate, an enzyme having a function of catalyzing the conversion of glyceraldehyde D-3-phosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, and an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose;

enzyme combination (IV-2-b): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate, a combination of an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into adenosine diphosphate- α -D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose into amylose;

enzyme combination (IV-2-c): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, a combination of an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, and an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose; or

Enzyme combination (IV-2-d): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into adenosine diphosphate-alpha-D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate into adenosine diphosphate A combination of enzymes that convert-alpha-D-glucose diphosphate to amylose;

the enzyme used in the step (c) is an enzyme with the function of catalyzing amylose to be converted into amylopectin.

8. A process (V) for the synthesis of starch, comprising the following steps:

the method comprises the following steps: formic acid is used as a raw material and is converted into formaldehyde under the catalysis of one or more enzymes;

step two: converting the formaldehyde obtained in the step I into dihydroxyacetone under the catalysis of one or more enzymes;

step three: secondly, the dihydroxyacetone obtained in the step two is converted into amylose under the catalysis of one or more enzymes; and

optional step (iv): the amylose obtained in the step three is converted into amylopectin under the catalysis of one or more enzymes;

preferably, the enzyme used in the step (i) is a single enzyme having the function of catalyzing the conversion of formic acid into formaldehyde; or a combination of enzymes from: enzyme combination (V-1-a): an enzyme having a function of catalyzing the conversion of formaldehyde into formyl-CoA with an enzyme having a function of catalyzing the conversion of formyl-CoA into formaldehyde, or a combination of enzymes (V-1-b): a combination of an enzyme having a function of catalyzing the conversion of formaldehyde into formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate into formyl coenzyme A, and an enzyme having a function of catalyzing the conversion of formyl coenzyme A into formaldehyde;

the enzyme used in the second step is an enzyme with the function of catalyzing formaldehyde to be converted into dihydroxyacetone;

the enzyme used in the third step is the following enzyme combination:

enzyme combination (V-3-a): a combination of an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into glyceraldehyde D-3-phosphate, an enzyme having a function of catalyzing the conversion of glyceraldehyde D-3-phosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, and an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose;

enzyme combination (V-3-b): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate, a combination of an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into adenosine diphosphate- α -D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose into amylose;

enzyme combination (V-3-c): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, a combination of an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, and an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose; or

Enzyme combination (V-3-d): an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate, an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-bisphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into adenosine diphosphate-alpha-D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate into adenosine diphosphate A combination of enzymes that convert-alpha-D-glucose diphosphate to amylose;

the enzyme used in the step (iv) is an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

9. A process (VI) for the synthesis of starch, comprising the following steps:

the method comprises the following steps: formic acid is used as a raw material and is converted into formaldehyde under the catalysis of one or more enzymes;

step two: converting the formaldehyde obtained in the step I into a compound F, namely D-3-glyceraldehyde phosphate under the catalysis of one or more enzymes;

step three: converting the obtained D-3-glyceraldehyde phosphate into a compound I, namely D-glucose-6-phosphate under the catalysis of one or more enzymes;

step IV: step three, converting the obtained D-glucose-6-phosphate into amylose under the catalysis of one or more enzymes; and

optional step (v): the amylose obtained in the step (iv) is converted into amylopectin under the catalysis of one or more enzymes;

preferably, the enzyme used in step (i) is a combination of enzymes (VI-1): a combination of an enzyme having a function of catalyzing the conversion of formate to formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate to formyl coenzyme A, and an enzyme having a function of catalyzing the conversion of formyl coenzyme A to formaldehyde;

the enzyme used in the step II is enzyme combination (VI-2): a combination of an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, and an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate;

the enzyme used in the third step is the following enzyme combination:

enzyme combination (VI-3-a): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate into D-fructose-1, 6-diphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate into D-fructose-6-phosphate, and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate; or

Enzyme combination (VI-3-b): a combination of an enzyme having a function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-6-phosphate and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate;

the enzyme used in the step (iv) is the following enzyme combination:

enzyme combination (VI-4-a): a combination of an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into adenosine diphosphate- α -D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose into amylose; or

Enzyme combination (VI-4-b): a combination of an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate and an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose;

the enzyme used in the fifth step is the enzyme with the function of catalyzing the amylose to be converted into the amylopectin.

10. The method of any one of claims 1-9, wherein each step, sub-step, or specific reaction of each method can be performed in steps, or any adjacent two, three, four, five, six, seven, or more steps, sub-steps, or specific reactions can also be performed simultaneously, or all steps or specific reactions can be performed simultaneously.

11. A synthetic method of dihydroxyacetone comprises the steps of converting methanol which is used as a raw material into dihydroxyacetone under the catalysis of one or more enzymes;

the steps include the steps of:

step (1): methanol is taken as a raw material and is converted into formaldehyde under the catalysis of enzyme with the function of catalyzing the conversion of the methanol into the formaldehyde; and

step (2): converting the formaldehyde obtained in the step (1) into dihydroxyacetone under the catalysis of an enzyme with the function of catalyzing the formaldehyde to be converted into the dihydroxyacetone;

preferably, the enzyme having a function of catalyzing the conversion of methanol into formaldehyde in step (1) includes, but is not limited to, Alcohol Oxidase (AOX) or a mutant thereof, cholesterol oxidase or a mutant thereof, Alcohol Dehydrogenase (ADH) or a mutant thereof, methanol dehydrogenase or a mutant thereof, L-threonine-3-dehydrogenase or a mutant thereof, cyclohexanol dehydrogenase or a mutant thereof, n-butanol dehydrogenase or a mutant thereof;

the enzyme having the function of catalyzing the conversion of formaldehyde into dihydroxyacetone in the step (2) includes, but is not limited to, Formaldehyde Lyase (FLS) or a mutant thereof (FLS-M), glycolaldehyde synthase (GALS) or a mutant thereof;

wherein, the steps (1) and (2) can be carried out synchronously or step by step; when the steps (1) and (2) are carried out synchronously, the reaction system comprises substrate methanol, the enzyme with the function of catalyzing the methanol to be converted into formaldehyde and the enzyme with the function of catalyzing the formaldehyde to be converted into dihydroxyacetone; optionally, a helper enzyme, such as catalase, may also be included.

Technical Field

The invention belongs to the technical field of biosynthesis, and particularly relates to a biosynthesis method of starch (including amylose and amylopectin).

Background

The global food production needs to consume 38% of land and 70% of fresh water resources, and with the increase of global population, the global food demand is predicted to be improved by 50-70% in 2050, and the current agricultural planting mode is difficult to meet the increasing food demand. Starch is an important food material, providing about half the energy required by the human body per day. In addition, starch is an important raw material for the biological production of chemicals, e.g., amino acids, organic acids, bioethanol, etc.

Farming is currently the only way to produce starch. The natural synthetic pathway of starch in crops involves the calvin cycle, involving a total of twenty-few chemical reactions and intermediary metabolites, as well as a number of organelles. Although starch synthesis is reported to be achieved, raw materials such as glucose-1-phosphate, glucose-adenosine diphosphate, sucrose, dextrin and the like are still obtained by agricultural planting, and therefore, the reported methods do not have the capability of producing starch instead of agricultural planting. The development of a method for producing starch instead of agricultural planting is of great significance.

Travelling into space and exploring the universe are always dream for human beings, but how to supply food under the space condition is always a great challenge. The aerospace country has been dedicated to developing food supply modes based on plant cultivation, but is limited by the mode of producing starch by crops, and dozens to hundreds of cubes of spaces are needed to meet the starch requirement of one person. And the development of a new starch synthesis route will help to achieve food supply in space conditions.

Although advances in metabolic engineering and synthetic biology have allowed the production of many pharmaceutical and agricultural products, such as artemisinin, opioids, lycopene, milk, and meat, by industrial fermentation, the industrial synthesis of starch has not been achieved.

Carbon dioxide is a major greenhouse gas and also an important carbon feedstock. About 100 hundred million tons of carbon dioxide are discharged every year in China, and can be used as an important industrial raw material to be converted and utilized. Efficient chemical catalysts have been developed at home and abroad, carbon dioxide can be reduced to generate simple compounds such as formic acid, methanol and the like by light, electricity and hydrogen energy, but the chemical catalysts cannot further synthesize complex compounds such as starch.

Therefore, there is a need in the art for a method of scientifically utilizing a carbon compound to reduce the emission of carbon dioxide, and for a method of biologically and industrially synthesizing starch. Currently, there is no technology or method that can meet both of these needs.

Disclosure of Invention

The invention provides a new way for synthesizing starch, which realizes the synthesis of amylose and/or amylopectin from a carbon compound through the combination of different chemical reactions.

The invention provides a method (I) for synthesizing starch, which comprises the steps of taking a compound D, namely dihydroxyacetone as a raw material, and converting the compound D into the starch under the catalysis of a plurality of enzymes;

the steps may include the steps of:

step (1): the compound D, namely dihydroxyacetone is used as a raw material and is converted into a compound F, namely D-3-glyceraldehyde phosphate under the catalysis of one or more enzymes;

step (2): converting the compound F obtained in the step (1) into a compound I, namely D-glucose-6-phosphate under the catalysis of one or more enzymes; and

and (3): converting the compound I obtained in the step (2) into starch under the catalysis of one or more enzymes.

According to an embodiment of process (I) of the present invention, the enzyme used in step (1) is an enzyme or a combination of enzymes which catalyze the conversion of dihydroxyacetone to D-3-glyceraldehyde phosphate via one or more reactions. For example, a combination of an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate to dihydroxyacetone phosphate and an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate to D-3-glyceraldehyde phosphate may be used.

According to an embodiment of process (I) of the invention, the enzyme used in step (2) is an enzyme or a combination of enzymes which catalyse the conversion of glyceraldehyde D-3-phosphate to D-glucose-6-phosphate via one or more reactions. For example, the following enzyme combinations are possible: enzyme combination (I-2-a): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate into D-fructose-1, 6-diphosphate, an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate into D-fructose-6-phosphate, and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate; or a combination of enzymes (I-2-b): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-3-phosphate to D-fructose-6-phosphate and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate to D-glucose-6-phosphate.

According to an embodiment of process (I) of the invention, the enzyme used in step (3) is an enzyme or a combination of enzymes which catalyse the conversion of D-glucose-6-phosphate to amylose or amylopectin by one or more reactions. For example, the following enzyme combinations are possible:

enzyme combination (I-3-a): a combination of an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into alpha-D-glucose-1-phosphate and an enzyme having a function of catalyzing the conversion of alpha-D-glucose-1-phosphate into amylose; or

Enzyme combination (I-3-b): a combination of an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into adenosine diphosphate- α -D-glucose, and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose into amylose;

optionally, the combination (I-3-a) or the combination (I-3-b) further comprises an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

According to an embodiment of process (I) of the invention, step (1) comprises the following sub-steps:

step (1-1): compound D is converted to compound E, dihydroxyacetone phosphate, catalyzed by one or more enzymes (this reaction is denoted as reaction 9); and

step (1-2): the compound E obtained in step (1-1) is converted into the compound F, i.e., D-3-glyceraldehyde phosphate, under the catalysis of one or more enzymes (this reaction is denoted as reaction 10).

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (1-1) is an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate.

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (1-2) is an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate to glyceraldehyde D-3-phosphate.

According to an embodiment of process (I) of the invention, step (2) comprises the following sub-steps:

step (2-1): compound F is converted to compound H, i.e. D-fructose-6-phosphate, under the catalysis of one or more enzymes; and

step (2-2): the compound H obtained in step (2-1) is converted into the compound I, namely D-glucose-6-phosphate, under the catalysis of one or more enzymes (this reaction is denoted as reaction 15).

According to an embodiment of process (I) of the present invention, the enzyme used in step (2-1) is an enzyme or a combination of enzymes that catalyzes the conversion of glyceraldehyde D-3-phosphate to D-fructose-6-phosphate by one or more reactions. For example, it may be an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate to D-fructose-6-phosphate alone, or it may be an enzyme combination (I-2-1): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate to D-fructose-1, 6-diphosphate and an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate to D-fructose-6-phosphate.

Specifically, step (2-1) may be performed as follows: compound F is converted to compound H (this reaction is referred to as reaction 13 or 14) by the catalysis of an enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate to D-fructose-6-phosphate. Where reactions 13 and 14 are carried out under catalysis of different enzymes, for example, the reaction in which glyceraldehyde D-3-phosphate is converted to D-fructose-6-phosphate by fructose-6-phosphate aldolase (FSA) or transaldolase is designated as reaction 13, and the reaction in which glyceraldehyde D-3-phosphate is converted to D-fructose-6-phosphate by fructose-6-phosphate aldolase phosphatase (FBAP) is designated as reaction 14.

Alternatively, the step (2-1) may be performed as follows: firstly, a compound F is converted into a compound G, namely D-fructose-1, 6-diphosphate (the reaction is marked as reaction 11) under the catalysis of an enzyme which has the function of catalyzing the conversion of D-3-glyceraldehyde phosphate into D-fructose-1, 6-diphosphate; then, the compound G is converted into D-fructose-6-phosphate by the action of an enzyme having a function of catalyzing the conversion of D-fructose-1, 6-diphosphate into D-fructose-6-phosphate (this reaction is referred to as reaction 12).

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (2-2) is an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate.

According to an embodiment of process (I) of the invention, step (3) comprises the following sub-steps:

step (3-1): compound I is converted to compound J, i.e. α -D-glucose-1-phosphate, under the catalysis of one or more enzymes (this reaction is denoted as reaction 16);

step (3-2): converting the compound J obtained in the step (3-1) into a compound 1, namely amylose, under the catalysis of one or more enzymes;

and optionally step (3-3): the compound 1 obtained in step (3-2) is converted into the compound 2, i.e., amylopectin, under the catalysis of one or more enzymes (this reaction is denoted as reaction 20).

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (3-1) is an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate.

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (3-2) is an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose. For example, it may be an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose alone, or it may be an enzyme combination (I-3-2): a combination of an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate to adenosine diphosphate- α -D-glucose and an enzyme having a function of catalyzing the conversion of adenosine diphosphate- α -D-glucose to amylose.

Specifically, step (3-2) may be performed as follows: compound J is converted to compound 1, amylose, by catalysis with an enzyme that catalyzes the conversion of alpha-D-glucose-1-phosphate to amylose (this reaction is designated as reaction 19). Alternatively, the step (3-2) may be performed as follows: firstly, compound J is converted to compound K, i.e., adenosine diphosphate- α -D-glucose, under the catalysis of an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate to adenosine diphosphate- α -D-glucose (this reaction is denoted as reaction 17); then, the obtained compound K is converted into amylose by the catalysis of an enzyme having a function of catalyzing the conversion of adenosine diphosphate-alpha-D-glucose into amylose (this reaction is referred to as reaction 18).

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (3-3) is an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

According to an embodiment of process (I) according to the invention, the process according to the invention also comprises, before step (1), a step (0) of converting formaldehyde into compound D, namely dihydroxyacetone, starting from formaldehyde, catalysed by one or more enzymes (this reaction is designated as reaction 8).

According to an embodiment of the process (I) of the present invention, the enzyme used in the step (0) is an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone.

According to an embodiment of process (I) of the invention, the process of the invention further comprises, before step (0), a step (a) of converting methanol or formic acid, starting from methanol, into formaldehyde, catalysed by one or more enzymes.

According to an embodiment of the process (I) of the present invention, in the step (a), when formaldehyde is synthesized from methanol as a raw material, the enzyme used is an enzyme having a function of catalyzing the conversion of methanol into formaldehyde.

According to an embodiment of the process (I) of the present invention, in the step (a), when formaldehyde is synthesized from formic acid as a starting material, the enzyme used is an enzyme having a function of catalyzing the conversion of formic acid into formaldehyde. For example, it may be an enzyme having a function of catalyzing the conversion of formic acid into formaldehyde alone, or it may be an enzyme combination (I-a-1): an enzyme having a function of catalyzing the conversion of formate to formyl-CoA with an enzyme having a function of catalyzing the conversion of formyl-CoA to formaldehyde, or a combination of enzymes (I-a-2): the combination of 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 coenzyme A, and an enzyme having a function of catalyzing the conversion of formyl coenzyme A to formaldehyde.

Specifically, in the step (a), when methanol is used as a raw material for synthesizing formaldehyde, the reaction may be carried out under the catalysis of alcohol oxidase or alcohol dehydrogenase. The reaction catalyzed by alcohol dehydrogenase is denoted as reaction 1, and the reaction catalyzed by alcohol oxidase is denoted as reaction 2.

Specifically, when formaldehyde is synthesized from formic acid as a raw material in step (a), the synthesis may be performed according to any one of the following steps (a1), (a2) or (a 3):

The invention also provides a process (II) for the synthesis of starch, comprising the steps of:

step 1): methanol is used as a raw material and is converted into amylose under the catalysis of a plurality of enzymes; and optionally step 2): converting the amylose obtained in the step 1) into amylopectin under the catalysis of one or more enzymes.

According to an embodiment of process (II) of the invention, the enzyme used in step 1) is a combination of enzymes which catalyse the synthesis of starch from methanol via a multi-step reaction. For example, the following enzyme combinations are possible:

Specifically, step 1) may be performed according to the above-described combination of reactions 2, 8, 9, 10, 13, 15, 16, 17 and 18, the combination of reactions 2, 8, 9, 10, 14, 15, 16, 17 and 18, the combination of reactions 2, 8, 9, 10, 11, 12, 15, 16, 17 and 18, the combination of reactions 2, 8, 9, 10, 13, 15, 16 and 19, the combination of reactions 2, 8, 9, 10, 14, 15, 16 and 19, or the combination of reactions 2, 8, 9, 10, 11, 12, 15, 16 and 19.

According to an embodiment of process (II) of the invention, the enzyme used in step 2) is an enzyme having the function of catalyzing the conversion of amylose into amylopectin.

Specifically, step 2) may be performed according to the above-described reaction 20.

The invention also provides a process (III) for the synthesis of starch, comprising the steps of:

step 1): methanol is used as a raw material and is converted into a compound D, namely dihydroxyacetone under the catalysis of one or more enzymes;

step 2): converting dihydroxyacetone obtained in the step 1) into amylose under the catalysis of one or more enzymes; and

optional step 3): converting the amylose obtained in the step 2) into amylopectin under the catalysis of one or more enzymes.

According to an embodiment of process (III) of the invention, the enzyme used in step 1) is the enzyme combination (III-1): a combination of an enzyme having the function of catalyzing the conversion of methanol to formaldehyde and an enzyme having the function of catalyzing the conversion of formaldehyde to dihydroxyacetone.

Specifically, step 1) may be performed according to the combination of reactions 1 and 8 or the combination of reactions 2 and 8 described above.

According to an embodiment of process (III) of the invention, the enzymes used in step 2) are the following enzyme combinations:

Specifically, step 2) may be performed according to the above-described combination of reactions 9, 10, 13, 15, 16, 17, and 18, the combination of reactions 9, 10, 14, 15, 16, 17, and 18, or the combination of reactions 9, 10, 11, 12, 15, 16, 17, and 18, the combination of reactions 9, 10, 13, 15, 16, and 19, the combination of reactions 9, 10, 14, 15, 16, and 19, or the combination of reactions 9, 10, 11, 12, 15, 16, and 19.

According to an embodiment of process (III) according to the invention, the enzyme used in step 3) is an enzyme having the function of catalyzing the conversion of amylose into amylopectin.

Specifically, step 3) may be performed according to the above-described reaction 20.

The invention also provides a process (IV) for the synthesis of starch, comprising the steps of:

the method comprises the following steps: formic acid is used as a raw material and is converted into dihydroxyacetone under the catalysis of one or more enzymes;

step two: converting dihydroxyacetone obtained in the step I into amylose under the catalysis of one or more enzymes; and

optional step (c): and secondly, converting the amylose obtained in the step II into amylopectin under the catalysis of one or more enzymes.

According to an embodiment of process (IV) of the present invention, the enzyme used in step (r) is an enzyme or a combination of enzymes that catalyze the conversion of formate to dihydroxyacetone via one or more reactions. For example, the following combinations of enzymes may be used:

Specifically, step (r) may be carried out according to the combination of reactions 3 and 8, the combination of reactions 4, 7 and 8 or the combination of reactions 5,6, 7 and 8 described above.

According to an embodiment of process (IV) of the invention, the enzyme used in step (II) is an enzyme or a combination of enzymes which catalyse the conversion of dihydroxyacetone to amylose in one or more reactions. For example, the following combinations of enzymes may be used:

Specifically, step (c) may be performed according to the above-described combination of reactions 9, 10, 13, 15, 16 and 19, the combination of reactions 9, 10, 14, 15, 16 and 19, the combination of reactions 9, 10, 13, 15, 16, 17 and 18, the combination of reactions 9, 10, 14, 15, 16, 17 and 18, the combination of reactions 9, 10, 11, 12, 15, 16 and 19, or the combination of reactions 9, 10, 11, 12, 15, 16, 17 and 18.

According to an embodiment of process (IV) of the present invention, the enzyme used in step (c) is an enzyme having the function of catalyzing the conversion of amylose into amylopectin.

Specifically, step (c) may be performed according to reaction 20 described above.

The invention also provides a process (V) for the synthesis of starch, comprising the following steps:

the method comprises the following steps: formic acid is used as a raw material and is converted into formaldehyde under the catalysis of one or more enzymes;

step two: converting the formaldehyde obtained in the step I into dihydroxyacetone under the catalysis of one or more enzymes;

step three: secondly, the dihydroxyacetone obtained in the step two is converted into amylose under the catalysis of one or more enzymes; and

optional step (iv): and c, converting the amylose obtained in the step c into amylopectin under the catalysis of one or more enzymes.

According to an embodiment of the process (V) of the present invention, the enzyme used in the step (I) is a separate enzyme having a function of catalyzing the conversion of formic acid into formaldehyde; or a combination of enzymes from: enzyme combination (V-1-a): an enzyme having a function of catalyzing the conversion of formaldehyde into formyl-CoA with an enzyme having a function of catalyzing the conversion of formyl-CoA into formaldehyde, or a combination of enzymes (V-1-b): the combination of an enzyme having a function of catalyzing the conversion of formaldehyde into formyl phosphate, an enzyme having a function of catalyzing the conversion of formyl phosphate into formyl coenzyme A, and an enzyme having a function of catalyzing the conversion of formyl coenzyme A into formaldehyde.

Specifically, step (r) may be carried out according to reaction 3, a combination of reactions 4 and 7, or a combination of reactions 5,6 and 7 as described above.

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

Specifically, step (c) may be performed according to the above-mentioned reaction 8.

According to an embodiment of method (V) of the invention, the enzymes used in step (c) are the following combinations of enzymes:

Specifically, step (c) may be performed according to the above-described combination of reactions 9, 10, 13, 15, 16, and 19, the combination of reactions 9, 10, 14, 15, 16, and 19, the combination of reactions 9, 10, 13, 15, 16, 17, and 18, the combination of reactions 9, 10, 14, 15, 16, 17, and 18, the combination of reactions 9, 10, 11, 12, 15, 16, and 19, or the combination of reactions 9, 10, 11, 12, 15, 16, 17, and 18.

According to an embodiment of the process (V) of the present invention, the enzyme used in the step (iv) is an enzyme having a function of catalyzing the conversion of amylose into amylopectin.

Specifically, step (iv) may be performed according to reaction 20 described above.

The invention also provides a process (VI) for the synthesis of starch, comprising the steps of:

the method comprises the following steps: formic acid is used as a raw material and is converted into formaldehyde under the catalysis of one or more enzymes;

step two: converting the formaldehyde obtained in the step I into a compound F, namely D-3-glyceraldehyde phosphate under the catalysis of one or more enzymes;

step three: converting the obtained D-3-glyceraldehyde phosphate into a compound I, namely D-glucose-6-phosphate under the catalysis of one or more enzymes;

step IV: step three, converting the obtained D-glucose-6-phosphate into amylose under the catalysis of one or more enzymes; and

optional step (v): and fourthly, converting the amylose obtained in the step IV into amylopectin under the catalysis of one or more enzymes.

According to an embodiment of process (VI) of the invention, the enzyme used in step (i) is a combination of enzymes (VI-1): a combination of an enzyme having a function of catalyzing the conversion of formate 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.

Specifically, step (r) may be carried out according to reaction 3, a combination of reactions 4 and 7, or a combination of reactions 5,6 and 7 as described above.

According to an embodiment of process (VI) of the invention, the enzyme used in step (II) is a combination of enzymes (VI-2): a combination of an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone, an enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate, and an enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate into D-3-glyceraldehyde phosphate.

Specifically, step (c) may be performed according to a combination of reactions 8, 9 and 10 described above.

According to an embodiment of method (VI) of the invention, the enzyme used in step (c) is a combination of enzymes:

Enzyme combination (VI-3-b): a combination of an enzyme having a function of catalyzing the conversion of glyceraldehyde-3-phosphate to D-fructose-6-phosphate and an enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate to D-glucose-6-phosphate.

Specifically, step (c) may be performed according to the combination of reactions 11, 12, and 15 described above, or step (c) may also be performed according to the combination of reactions 13 and 15 described above, or the combination of reactions 14 and 15.

According to an embodiment of process (VI) of the invention, the enzymes used in step (iv) are the following combinations of enzymes:

Specifically, step iv may be performed according to a combination of reactions 16, 17, and 18 described above, or step iv may also be performed according to a combination of reactions 16 and 19 described above.

According to an embodiment of process (VI) according to the invention, the enzyme used in step (v) is an enzyme having the function of catalyzing the conversion of amylose to amylopectin.

Specifically, step (c) may be performed according to the reaction 20 described above.

The invention also provides a method for synthesizing dihydroxyacetone, which comprises the step of converting methanol serving as a raw material into dihydroxyacetone under the catalysis of one or more enzymes.

According to an embodiment of the present invention, the method for synthesizing dihydroxyacetone comprises the steps of:

step (2): and (2) converting the formaldehyde obtained in the step (1) into dihydroxyacetone under the catalysis of an enzyme with the function of catalyzing the formaldehyde to be converted into the dihydroxyacetone.

According to an embodiment of the present invention, the enzyme having a function of catalyzing the conversion of methanol into formaldehyde in step (1) includes, but is not limited to, Alcohol Oxidase (AOX) or a mutant thereof, cholesterol oxidase or a mutant thereof, Alcohol Dehydrogenase (ADH) or a mutant thereof, methanol dehydrogenase or a mutant thereof, L-threonine-3-dehydrogenase or a mutant thereof, cyclohexanol dehydrogenase or a mutant thereof, n-butanol dehydrogenase or a mutant thereof.

The enzyme having the function of catalyzing the conversion of formaldehyde into dihydroxyacetone in the step (2) includes, but is not limited to, Formaldehyde Lyase (FLS) or a mutant thereof (FLS-M), glycolaldehyde synthase (GALS) or a mutant thereof.

According to an embodiment of the present invention, steps (1) and (2) may be performed simultaneously or in steps.

According to an embodiment of the present invention, steps (1) and (2) are performed simultaneously, for example, the reaction system comprises a substrate of methanol, an enzyme having a function of catalyzing the conversion of methanol into formaldehyde, and an enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone. For example, the reaction system contains substrates methanol, alcohol oxidase or alcohol dehydrogenase, and formaldehyde lyase. In addition, the reaction system may optionally contain a helper enzyme such as catalase.

According to an embodiment of the invention, the reaction system further comprises NaCl, Mg²⁺、Zn²⁺And the like.

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 the present invention, each step, sub-step and specific reaction (such as reaction 1, reaction 2, etc., wherein the reaction numbers refer to the above summary or attached figure 1) of each method of the present invention can be performed step by step, or any adjacent two, three, four, five, six, seven or more steps, sub-steps or specific reactions can be performed simultaneously, or all steps or specific reactions can be performed simultaneously. By "adjacent" steps, sub-steps or particular reactions, it is meant that the product of a previous step, sub-step or particular reaction can be used as a reactant for a subsequent step, sub-step or particular reaction, and the two steps, sub-steps or particular reactions can be referred to as "adjacent". The step-by-step reaction refers to that after the previous step reaction is finished, the product is purified or not, and then enzyme or required components for catalyzing the next step reaction are added for the next step reaction; 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. For example, the reactions attached as "-" below indicate that they are performed simultaneously: reaction 2-8, reaction 4-7, reaction 5-6-7-8, reaction 8-9-10, reaction 11-12-15, reaction 13-15, reaction 14-15, reaction 16-17-18, reaction 16-19, reaction 9-10-11-12-15-16-17-18, reaction 9-10-13-15-16-17-18, reaction 9-10-14-15-16-17-18, reaction 9-10-13-15-16-19, reaction 9-10-11-12-15-16-17-20, reaction 2-8-9-10-11-12-15-16-17- 18. For example, "reactions 2-8" means that reaction 2 and reaction 8 are carried out simultaneously; "reactions 4-7" means that reaction 4 and reaction 7 are carried out simultaneously, and so on.

In the context of the present invention, "combination of reactions a and b. For example, "a combination of reactions 4 and 7" means that the conversion of formic acid to formaldehyde is achieved by carrying out reaction 4 and reaction 7, wherein reaction 4 and reaction 7 can be carried out separately or simultaneously; "combination of reactions 9, 10, 11, 12, 15, 16 and 19" means that the conversion of dihydroxyacetone to amylose is achieved by carrying out reactions 9, 10, 11, 12, 15, 16 and 19, wherein adjacent two, three, four, five, six or seven of reactions 9, 10, 11, 12, 15, 16 and 19 can be carried out stepwise or simultaneously; and so on.

According to the invention, the different reactions in which the starting materials and the products are identical in the same step or in sub-steps of the respective process can be interchanged.

According to the invention, the reactions in the different steps or sub-steps of the respective processes can be combined at will, as long as the latter reaction is starting from the product of the former reaction and their combination enables the synthesis of the final product starch.

For example, compound 1 (i.e., amylose) can be achieved by a combination of the following reactions starting from dihydroxyacetone (see figure 1 for reaction numbers): combinations of reactions 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 9, 10, 13, 15, 16, 17, and 18, combinations of reactions 9, 10, 14, 15, 16, 17, and 18, combinations of reactions 9, 10, 11, 12, 15, 16, and 19, combinations of reactions 9, 10, 13, 15, 16, and 19, and combinations of reactions 9, 10, 14, 15, 16, and 19.

Compound 2 (i.e. amylopectin) can be achieved by a combination of the following reactions starting from dihydroxyacetone (see figure 1 for reaction numbers): combinations of reactions 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 9, 10, 13, 15, 16, 17, 18, and 20, combinations of reactions 9, 10, 14, 15, 16, 17, 18, and 20, combinations of reactions 9, 10, 11, 12, 15, 16, 19, and 20, combinations of reactions 9, 10, 13, 15, 16, 19, and 20, and combinations of reactions 9, 10, 14, 15, 16, 19, and 20.

Compound 1 (i.e. amylose) can be achieved from formaldehyde by a combination of the following reactions (see figure 1 for reaction numbers): combinations of reactions 8, 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 8, 9, 10, 13, 15, 16, 17, and 18, combinations of reactions 8, 9, 10, 14, 15, 16, 17, and 18, combinations of reactions 8, 9, 10, 11, 12, 15, 16, and 19, combinations of reactions 8, 9, 10, 13, 15, 16, and 19, and combinations of reactions 8, 9, 10, 14, 15, 16, and 19.

Compound 2 (i.e. amylopectin) can be achieved starting from formaldehyde by a combination of the following reactions (see figure 1 for reaction numbers): combinations of reactions 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 8, 9, 10, 13, 15, 16, 17, 18, and 20, combinations of reactions 8, 9, 10, 14, 15, 16, 17, 18, and 20, combinations of reactions 8, 9, 10, 11, 12, 15, 16, 19, and 20, combinations of reactions 8, 9, 10, 13, 15, 16, 19, and 20, and combinations of reactions 8, 9, 10, 14, 15, 16, 19, and 20.

Compound 1 (i.e. amylose) can be achieved from methanol, formic acid as starting materials by a combination of the following reactions (see figure 1 for reaction numbers): combinations of reactions 1, 8, 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 2, 8, 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 3, 8, 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 4, 7, 8, 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 5,6, 7, 8, 9, 10, 11, 12, 15, 16, 17, and 18, combinations of reactions 1, 8, 9, 10, 13, 15, 16, 17, and 18, combinations of reactions 2, 8, 9, 10, 13, 15, 16, 17, and 18, combinations of reactions 3, 8, 9, 10, 13, 15, 16, 17, and 18, combinations of reactions 4, 7, 8, 9, 10, 13, 15, 16, 17, and 18, combinations of reactions 5,6, 7, 8, 9, 15, 16, 17, and 18, combinations of reactions 5,6, 8, 9, 10, 15, 17, and 18, 9. 10, 14, 15, 16, 17 and 18, reactions 2, 8, 9, 10, 14, 15, 16, 17 and 18, reactions 3, 8, 9, 10, 14, 15, 16, 17 and 18, reactions 4, 7, 8, 9, 10, 14, 15, 16, 17 and 18, reactions 5,6, 7, 8, 9, 10, 14, 15, 16, 17 and 18, reactions 1, 8, 9, 10, 11, 12, 15, 16 and 19, reactions 2, 8, 9, 10, 11, 12, 15, 16 and 19, reactions 3, 8, 9, 10, 11, 12, 15, 16 and 19, reactions 4, 7, 8, 9, 10, 12, 15, 16 and 19, reactions 5,6, 7, 8, 9, 10, 11, 12, 15, 16 and 19, reactions 1, 8, 9, 13, 15, 19, combinations of reactions 2, 8, 9, 10, 13, 15, 16, and 19, combinations of reactions 3, 8, 9, 10, 13, 15, 16, and 19, combinations of reactions 4, 7, 8, 9, 10, 13, 15, 16, and 19, combinations of reactions 5,6, 7, 8, 9, 10, 13, 15, 16, and 19, combinations of reactions 1, 8, 9, 10, 14, 15, 16, and 19, combinations of reactions 2, 8, 9, 10, 14, 15, 16, and 19, combinations of reactions 3, 8, 9, 10, 14, 15, 16, and 19, combinations of reactions 4, 7, 8, 9, 10, 14, 15, 16, and 19, and combinations of reactions 5,6, 7, 8, 9, 10, 14, 15, 16, and 19.

Compound 2 (i.e. amylopectin) can be achieved from methanol, formic acid as starting materials by a combination of the following reactions (see figure 1 for reaction numbers): combinations of reactions 1, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 2, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 3, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 4, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 5,6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 20, combinations of reactions 1, 8, 9, 10, 13, 15, 16, 17, 18, and 20, combinations of reactions 2, 8, 9, 10, 13, 15, 16, 17, 18, and 20, combinations of reactions 3, 8, 9, 10, 13, 15, 16, 17, 18, and 20, combinations of reactions 4, 7, 8, 9, 10, 13, 15, 17, 18, and 20, combinations of reactions 4, 8, 9, 10, 13, 15, 17, 18, and 20, combinations of reactions 3, 6, 9, 6, 5, 9, 20, 13. 15, 16, 17, 18, and 20, reaction 1, 8, 9, 10, 14, 15, 16, 17, 18, and 20, reaction 2, 8, 9, 10, 14, 15, 16, 17, 18, and 20, reaction 3, 8, 9, 10, 14, 15, 16, 17, 18, and 20, reaction 4, 7, 8, 9, 10, 14, 15, 16, 17, 18, and 20, reaction 5,6, 7, 8, 9, 10, 14, 15, 16, 17, 18, and 20, reaction 1, 8, 9, 10, 11, 12, 15, 16, 19, and 20, reaction 2, 8, 9, 10, 11, 12, 15, 16, 19, and 20, reaction 3, 8, 9, 10, 11, 12, 15, 16, 19, and 20, reaction 4, 7, 8, 9, 10, 11, 12, 15, 16, 19, and 20, reaction 5,6, 7, 5, 7, 20, 9. 10, 11, 12, 15, 16, 19, and 20, reaction 1, 8, 9, 10, 13, 15, 16, 19, and 20, reaction 2, 8, 9, 10, 13, 15, 16, 19, and 20, reaction 3, 8, 9, 10, 13, 15, 16, 19, and 20, reaction 4, 7, 8, 9, 10, 13, 15, 16, 19, and 20, reaction 5,6, 7, 8, 9, 10, 13, 15, 16, 19, and 20, reaction 1, 8, 9, 10, 14, 15, 16, 19, and 20, reaction 2, 8, 9, 10, 14, 15, 16, 19, and 20, reaction 3, 8, 9, 10, 14, 15, 16, 19, and 20, reaction 4, 7, 8, 9, 10, 14, 15, 16, 19, and 20, and combinations of reactions 5,6, 7, 8, 9, 10, 14, 15, 16, 19, and 20.

The enzyme used in the context of the present application when the expression "enzyme having a function of catalyzing the conversion of a substance a into a substance B" is used herein 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, the term "enzyme having a function of catalyzing the conversion of methanol into formaldehyde" refers to an enzyme that can catalyze a reaction of converting methanol into formaldehyde, and includes, but is not limited to, alcohol oxidase (AOX, EC 1.1.3.13) or a mutant thereof, cholesterol oxidase (EC 1.1.3.6) or a mutant thereof, alcohol dehydrogenase (ADH, EC 1.1.1.1.1; EC 1.1.2; EC 1.1.1.2; EC 1.1.1.71; EC 1.1.2.8) or a mutant thereof, methanol dehydrogenase (EC 1.1.1.1.244; EC 1.1.2.7; EC 1.2.B2) or a mutant thereof, L-threonine-3-dehydrogenase (EC 1.1.1.103) or a mutant thereof, cyclohexanol dehydrogenase (EC 1.1.1.245 or a mutant thereof), n-butanol dehydrogenase (EC 1.1.2.9) or a mutant thereof, and these enzymes may be derived from, but not limited to, different species such as pichia pastoris, candida spp, streptomyces, corynebacterium glutamicum, escherichia coli, rhodococcus, and ceramic. "an enzyme having a function of catalyzing the conversion of formic acid into formaldehyde" refers to an enzyme that can catalyze the reaction of converting formic acid into formaldehyde, and includes, but is not limited to, aldehyde dehydrogenase (ADH, EC 1.2.1.3; EC 1.2.1.4; EC 1.2.1.5) or a mutant thereof, and these enzymes can be derived from, but not limited to, Burkholderia, Pseudomonas, Acetobacter, yeast, and the like, and various species. "enzyme having a function of catalyzing the conversion of formate to formyl-CoA" refers to an enzyme that can catalyze the reaction of converting formate to formyl-CoA, including but not limited to acetyl-CoA synthetase (ACS, EC 6.2.1.1) or a mutant thereof, which can be derived from, but not limited to, Escherichia coli, Salmonella, Pseudomonas, Mueller, Thermus, etc., 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) or a mutant thereof, which can be derived from, but not limited to, listeria, pseudomonas, acinetobacter, giardia, and the like, of various species. "enzyme having a function of catalyzing the conversion of formate to formyl phosphate" refers to an enzyme that can catalyze the conversion of formate to formyl phosphate, including but not limited to acetate kinase (ACKA, EC 2.7.2.1) or a mutant thereof, 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 coenzyme A" refers to an enzyme that can catalyze the reaction of converting formyl phosphate to formyl coenzyme A, including but not limited to, phosphate acetyltransferase (PTA, EC 2.3.1.8) or a mutant thereof, which can be derived from, but not limited to, Escherichia coli, Clostridium, Thermotoga, Methanosarcina, and the like, of various species. "enzyme having a function of catalyzing the conversion of formaldehyde into dihydroxyacetone" refers to an enzyme that can catalyze a reaction of converting formaldehyde into dihydroxyacetone, and includes, but is not limited to, Formaldehyde Lyase (FLS) or a mutant thereof (FLS-M), glycolaldehyde synthase (GALS) or a mutant thereof. "enzyme having a function of catalyzing the conversion of dihydroxyacetone into dihydroxyacetone phosphate" refers to an enzyme that can catalyze the reaction of converting dihydroxyacetone into dihydroxyacetone phosphate, and includes, but is not limited to, triose kinase (EC 2.7.1.28) or a mutant thereof, hydroxyacetone kinase (DAK, EC 2.7.1.29) or a mutant thereof, glycerol kinase (EC 2.7.1.30) or a mutant thereof, and these enzymes can be derived from, but are not limited to, Escherichia coli, Clostridium, Saccharomyces cerevisiae, Pichia pastoris, Citrobacter, Bacillus thermophilus, human, and the like. "enzyme having a function of catalyzing the conversion of dihydroxyacetone phosphate to D-3-glyceraldehyde phosphate" refers to an enzyme that can catalyze the reaction of converting dihydroxyacetone phosphate to D-3-glyceraldehyde phosphate, and includes, but is not limited to, triosephosphate isomerase (TPI, EC5.3.1.1) or a mutant thereof, which may be derived from, but is not limited to, Escherichia coli, yeast, Staphylococcus aureus, Mycobacterium tuberculosis, Arabidopsis thaliana, human, and the like.

"enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate into D-fructose-1, 6-diphosphate" refers to an enzyme that can catalyze the reaction of glyceraldehyde-D-3-phosphate and dihydroxyacetone phosphate into D-fructose-1, 6-diphosphate, including but not limited to fructose-diphosphate aldolase (FBA, EC 4.1.2.13) or a mutant thereof, which can be derived from, but not limited to, Escherichia coli, yeast, Bacillus, algae, Clostridium, human, and the like, and various species. "enzyme having a function of catalyzing the conversion of D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate" refers to an enzyme that can catalyze the reaction of converting D-fructose-1, 6-bisphosphate into D-fructose-6-phosphate, and includes, but is not limited to, fructose-bisphosphatase (FBP, EC 3.1.3.11) or a mutant thereof (FBP-M), inositol phosphatase (EC 3.1.3.25) or a mutant thereof, and N-acylneuraminic acid-9-phosphatase (EC 3.1.3.29) or a mutant thereof, and these enzymes may be derived from, but not limited to, Escherichia coli, Corynebacterium glutamicum, yeast, Bacillus, algae, Clostridium, human, and the like, and various species. "enzyme having a function of catalyzing the conversion of glyceraldehyde-D-3-phosphate into D-fructose-6-phosphate" refers to an enzyme that can catalyze the reaction of glyceraldehyde-D-3-phosphate and dihydroxyacetone or dihydroxyacetone phosphate into D-fructose-6-phosphate, and includes, but is not limited to, fructose-6-phosphate aldolase (FSA) or a mutant thereof, transaldolase (EC 2.2.1.2) or a mutant thereof, fructose-6-phosphate aldolase phosphatase (FBAP, EC 4.1.2.13&3.1.3.11) or a mutant thereof, which can be derived from, but not limited to, Escherichia coli, yeast, algae, archaea, human, and the like, and various species thereof. "enzyme having a function of catalyzing the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate" refers to an enzyme that can catalyze the conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, and includes, but is not limited to, phosphoglucoisomerase (PGI, EC 5.3.1.9) or a mutant thereof, phosphomannose isomerase (EC 5.3.1.8) or a mutant thereof, and these enzymes may be derived from, but not limited to, Escherichia coli, yeast, methanogen, Mycobacterium tuberculosis, Salmonella, Rhizobium, and the like. "an enzyme having a function of catalyzing the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate" refers to an enzyme that can catalyze the conversion of D-glucose-6-phosphate into α -D-glucose-1-phosphate, and includes, but is not limited to, hexophosphate mutase (PGM, EC5.4.2.2) or a mutant thereof, and acetylglucosamine phosphate mutase (EC 5.4.2.3) or a mutant thereof, and these enzymes can be derived from, but are not limited to, Escherichia coli, lactococcus lactis, yeast, Bacillus, Pseudomonas, Salmonella, maize, human, and other different species. "an enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate to ADP- α -D-glucose" refers to an enzyme that can catalyze the reaction of converting α -D-glucose-1-phosphate to ADP- α -D-glucose, including, but not limited to, glucose-1-phosphate adenylyl transferase (GlgC, EC 2.7.7.27) or a mutant thereof, which may be derived from, but not limited to, Escherichia coli, lactococcus lactis, yeast, Bacillus, Pseudomonas, Agrobacterium tumefaciens, maize, potato, and other different species. "enzyme having a function of catalyzing the conversion of adenosine diphosphate-alpha-D-glucose into amylose" refers to an enzyme that can catalyze the reaction of converting adenosine diphosphate-alpha-D-glucose into amylose, including but not limited to starch synthase (GlgA, EC 2.4.1.21) or a mutant thereof, which may be derived from, but not limited to, Escherichia coli, yeast, Bacillus, algae, corn, potato, and the like, among various species. "enzyme having a function of catalyzing the conversion of α -D-glucose-1-phosphate into amylose" refers to an enzyme that can catalyze the reaction of converting α -D-glucose-1-phosphate into amylose, including, but not limited to, starch phosphorylase (α GP, EC 2.4.1.1) alone or a mutant thereof, or a combination of glucose-1-phosphate adenylyl transferase (GlgC, EC 2.7.7.27) or a mutant thereof and starch synthase (GlgA, EC 2.4.1.21) or a mutant thereof, which may be derived from, but not limited to, e.coli, yeast, bacillus, algae, corn, potato, and the like, or a mutant thereof. "enzyme having a function of catalyzing the conversion of amylose into amylopectin" refers to an enzyme that can catalyze the reaction of converting amylose into amylopectin, including, but not limited to, 1,4- α -D-glucan branching enzyme (GlgB, EC 2.4.1.18) or a mutant thereof, which may be derived from, but not limited to, escherichia coli, bacillus, vibrio, yeast, corn, potato, and other various species.

The enzyme or mutant thereof used in the context of the present invention may be in the form of crude enzyme solution, crude enzyme solution lyophilized powder, pure enzyme 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 or the mutant thereof 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 (3) expressing the enzyme or the mutant thereof in a host cell to obtain a recombinant cell, namely the whole cell.

Advantageous effects

The method can realize the artificial biosynthesis of starch from simple compounds such as dihydroxyacetone, formaldehyde, formic acid, methanol and the like, and can even realize the total artificial biosynthesis of starch by coupling with methods such as carbon dioxide chemical reduction and the like which are mature at present and even taking carbon dioxide as a starting material. The synthesis of the natural starch needs Karlvin circulation, 21-22 steps of reaction are needed, the method only needs 9-12 steps of reaction, nearly half of reaction steps are reduced, the method does not need a recognized rate-limiting enzyme Rubisco, the synthesis speed is more advantageous, and the synthesis period can be greatly shortened. In addition, compared with the method that only low-concentration carbon dioxide and low-energy-density solar energy in the air can be utilized in agricultural planting, the method can utilize high-concentration carbon dioxide and high-energy-density electric energy and hydrogen energy, is more suitable for an industrial production mode, can shorten the production period from a plurality of months of agricultural planting to a plurality of days, is expected to greatly shorten agricultural land and water, and has important significance for solving the problems of large population, less cultivated land and limited fresh water resources in China. In addition, the characteristics also enable the method of the invention to be suitable for realizing the circular supply of starch in closed spaces such as spacecrafts, space stations and the like, and have important significance for space exploration strategies in China.

Drawings

Fig. 1 shows a synthetic route for the synthesis of compound 1, amylose, and compound 2, amylopectin, starting from methanol or formic acid.

Figure 2 shows a schematic of the structure of compound 1.

Figure 3 shows a schematic of the structure of compound 2.

Figure 4 shows the yield of the synthesis route from methanol to compound D (i.e. dihydroxyacetone).

Fig. 5 shows full wavelength scan spectra before and after the pathway reaction from compound 1 (i.e., amylose) to compound 2 (amylopectin).

Figure 6 shows the yields of different routes from compound D to compound 1.

Figure 7 shows the yields of different routes from compound D to compound 2.

Figure 8 shows the yield of the synthetic pathway from methanol to compound 1.

Detailed Description

The synthesis method of the present invention will be described in further detail with reference to specific 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.

Unless otherwise specified, the catalysts (enzymes) used in the reactions of the examples are shown in table 1 below:

table 1 catalyst used in each reaction according to each example

#: FLS gene information is derived from the literature references (Siegel, J.B., et al., computerized protein design enzymes a novel one-carbon architectural pathway, Proc Natl Acad Sci U S A,2015.112(12): p.3704-9.)

Alcohol dehydrogenase and alcohol oxidase were purchased from Sigma, Inc. (https:// www.sigmaaldrich.com/china-mainland. html). Aldehyde dehydrogenase, acetyl-coenzyme A synthetase, acetate kinase, phosphate acetyltransferase, acetaldehyde dehydrogenase, catalase, formate dehydrogenase, inorganic pyrophosphatase are obtained by means of PCR or gene synthesis and are cloned into corresponding expression vectors pET 28-a-pET 21-b-ACDS, pET 28-49323-ACDS, pET 28-15-ACDS, PTA-17-PTA 20-b, FADT 21-b, pET 26-b and pET 28-a vectors (Novagen, MaACDN, Wis.) by a Simple Cloning method (Simple Cloning) (You, C., et al (2012) 'Simple Cloning via Direct Transformation of PCR products and Bacillus subtilis)' 1593-1595 }, respectively. All eight plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

Formaldehyde lyase, hydroxyacetone kinase and triose phosphate isomerase are obtained by means of PCR or gene synthesis and are cloned into pET21a, pET21b and pET28a vectors (Novagen, Madison, Wis.) respectively by a simple cloning method (You, C., et al. (2012). appl. environ. Microbiol.78(5):1593-1595.), so as to obtain corresponding expression vectors pET21a-FLS, pET21b-TPI and pET28 a-DAK. All three plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

Fructose-bisphosphate aldolase, fructose-bisphosphatase, fructose-6-phosphate aldolase phosphatase, and glucose phosphate isomerase were obtained by PCR or gene synthesis, and cloned into pET21b vector (Novagen, Madison, Wis.) by simple cloning (You, C., et al (2012), appl. environ. Microbiol.78(5): 1593-1595), respectively, to obtain the corresponding expression vectors pET21b-FBA, pET21b-FBP, pET21b-FSA, pET21b-FBAP, pET21 b-PGI. All five plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

Hexose phosphate mutase, glucose-1-phosphate adenylyl transferase, starch synthase and starch phosphorylase are obtained by means of PCR or gene synthesis and cloned into pET20b and pET21b vectors (Novagen, Madison, Wis.) respectively by a simple cloning method (You, C., et al. (2012). appl. environ. Microbiol.78(5):1593-1595.) to obtain the corresponding expression vectors pET21b-PGM, pET21b-GlgC, pET21b-GlgA and pET20 b-alpha GP. All four plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

The 1, 4-alpha-D-glucan branching enzyme is obtained by means of PCR or gene synthesis and is cloned into pET28a vector (Novagen, Madison, Wis.) respectively by a simple cloning method (You, C., et al. (2012). Appl. environ. Microbiol.78(5):1593-1595.), so as to obtain the corresponding expression vector pET28 a-GlgB. This plasmid was transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

EXAMPLE 1 Synthesis of Compound C, Formaldehyde, from formic acid or methanol

There are five routes to the conversion of formic acid or methanol to compound C that can be achieved: route 1 (from methanol to formaldehyde), route 2 (from methanol to formaldehyde), route 3 (from formic acid to formaldehyde), route 4-7 (from formic acid to formyl-coa to formaldehyde), and route 5-6-7 (from formic acid to formyl-phosphate, to formyl-coa to formaldehyde) (see figure 1 for reaction numbers). First, a catalyst (i.e., an enzyme) that can catalyze each chemical reaction of each pathway was selected (see table 1), but the enzyme having a corresponding catalytic function is not limited to those listed in table 1. Then combining different catalysts according to a path to establish a corresponding reaction system, and detecting the yield of the formaldehyde after reacting for a period of time.

The yield of compound C 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, centrifuging to obtain 200. mu.L of a supernatant, detecting the OD414 value, and calculating the content of the compound C according to a formaldehyde standard curve.

Route 1: the reaction system is Tris buffer solution 100mM, NaCl 100mM and Mg with pH8.5²⁺5mM，Zn²⁺10μM，ADH 3.5mg/mL，NAD⁺100mM, methanol 1M; the reaction was carried out for 3 hours, and the yield of formaldehyde was 0.27 mM.

Route 2: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, AOX 1U/mL, CAT 300U/mL (helper enzyme, for eliminating AOX-produced hydrogen peroxide), methanol 20 mM; the reaction was carried out for 0.5 hour with a formaldehyde yield of 12 mM.

Route 3: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, FADH 4mg/mL, NADH 100mM, sodium formate 250mM, reaction for 3 hours, formaldehyde yield 0.1 mM.

Path 4-7: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, ATP 2mM, NADH 1.5mM, CoA 0.1mM, ACS 3.7mg/mL, ACDH 0.2mg/mL, FDH 0.024mg/mL (helper enzyme for regenerating NADH), PPase 0.1mg/mL (helper enzyme for hydrolyzing pyrophosphate), sodium formate 50mM, reaction 1 hour, formaldehyde yield 0.6 mM.

Paths 5-6-7: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, NADH 0.5mM, ATP 10mM, CoA 0.1mM, β -mercaptoethanol 2mM, ACKA 0.24mg/mL, PTA 1.2mg/mL, ACDH 0.2mg/mL, FDH 0.024mg/mL (auxiliary enzyme,for regeneration of NADH), sodium formate 50 mM; the reaction was carried out for 1 hour, and the yield of formaldehyde was 2 mM.

Example 2 Synthesis of Compound D, dihydroxyacetone, from methanol

The synthesis of compound D from methanol can be achieved by the following route:

path 2-8: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, AOX 1U/mL, CAT 300U/mL, methanol 20mM, FLS 5 mg/mL; the reaction time was 2 hours, and the yield of Compound D was 2.1 mM.

Detection method of compound D: 5mM dilute sulfuric acid as mobile phase, HX87 column (Bio-Rad, Aminex @ 300 mM. times.78 mM), flow rate of 0.6mL/min, loading 10 μ L/needle, according to DHA standard curve calculation of compound D yield. The yields of pathways 2-8 from methanol to compound D are shown in figure 4.

Example 3 Synthesis of Compound D, dihydroxyacetone, from Formaldehyde

The synthesis of compound D from formaldehyde can be achieved by the following route:

path 8: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, formaldehyde 25mM, TPP 0.5mM, FLS (source: Pseudomonas putida)10 mg/mL; after a reaction time of 2 hours, compound D was detected by the method of example 2, and as a result: the yield of compound D synthesized using formaldehyde lyase FLS was 7.092 mM.

Example 4 Synthesis of Compound D, dihydroxyacetone, from formic acid

The synthesis of compound D from formaldehyde can be achieved by the following route:

path 5-6-7-8: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, 50mM sodium formate, 0.5mM TPP, 0.1mM CoA, 10mM ATP, 2mM β -mercaptoethanol, 0.24mg/mL ACKA, 1.2mg/mL PTA, 0.2mg/mL ACDH, 0.024mg/mL FDH, 0.5mM NADH, 7.5mg/mL FLS (source: Pseudomonas putida) was added after 1-1.5 hours of reaction at 30 ℃ and after 3.5 hours of further reaction at 30 ℃, Compound D was detected by gas-mass spectrometry, resulting in a yield of 0.23mM of Compound D synthesized in pathway 5-6-7-8.

The gas-mass spectrometry detection method of compound D is as follows:

1) sample pretreatment, 500. mu.L of an appropriately diluted sample, 100. mu.L of 200mM PFBOA (O- (2,3,4,5,6-Pentafluorobenzyl) hydroxamine hydrochloride, O- (2,3,4,5,6-Pentafluorobenzyl) hydroxylamine hydrochloride), reaction at 30 ℃ for 1 hour, extraction with 100. mu.L of N-hexane, addition of 40. mu.L of an organic phase to an equal volume of MSTFA (N-Methyl-N- (trimethylsilyl) trifluoracetamide), reaction at 37 ℃ for 2 hours.

2) Gas chromatography conditions: an Agilent 7890A gas chromatograph, wherein a carrier gas is helium, the flow rate of the carrier gas is 1.2mL/min, a chromatographic column is a DB-5MS Ultra insert capillary column (30m multiplied by 250 mu m multiplied by 0.25 mu m), the initial temperature is 60 ℃, the initial temperature is kept for 1 minute, the operation is carried out for 1 minute, the heating rate is 1, 5 ℃/min to 240 ℃, the operation is carried out for 9 minutes, the heating rate is 2, 25 ℃/min to 300 ℃, the operation is carried out for 5 minutes, the operation is carried out for 22 minutes, the sample injection amount is 1 mu L/needle, and the sample injection port temperature is 250 ℃.

3) Mass spectrum conditions: agilent 7200Q-TOF mass spectrometer with solvent delay set for 4 minutes, power mode EI, electron energy 70eV, ion source temperature: 230 ℃, scan range: 35-550amu, mass spectrometry acquisition rate 5 spectra/s.

4) Data Analysis, using Mass Hunter software Qualitive Analysis, target metabolites were determined by NIST chemical database alignment and artificial Analysis, and DHA yield was calculated.

EXAMPLE 5 Synthesis of Compound C, i.e., Formaldehyde, to Compound F, i.e., glyceraldehyde D-3-phosphate

The conversion from compound C to compound F can be achieved by pathways 8-9-10 (see FIG. 1 for reaction numbers). First, a catalyst capable of catalyzing each chemical reaction in the pathway was selected (see Table 1), but the enzyme having the catalytic function is not limited to those listed in Table 1. Then combining different catalysts according to a path to establish a corresponding reaction system, and detecting the yield of the compound F after reacting for a period of time.

The compound E and the compound F are isomers, but the accumulation amount of the compound F is low due to unfavorable free energy, and thus the production amount of the compound E + the compound F is measured. Chemical combinationThe yield of compound E + compound F was determined as follows: 100 μ L of the assay system included 100mM Hepes buffer, pH7.5, 100mM NaCl, 20U/mL glyceraldehyde phosphate dehydrogenase, 20U/mL triose phosphate isomerase, 1mM NAD⁺4mM potassium arsenate; after the reaction is terminated, a suitably diluted sample to be tested is taken and the change in OD340 is detected. The yield of compound E + compound F was calculated according to a standard curve for glyceraldehyde 3-phosphate.

Paths 8-9-10: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, ATP 10mM, thiamine pyrophosphate 0.5mM, FLS 5mg/mL, DAK 0.1mg/mL, TPI 0.14mg/mL, Compound C10 mM; the reaction time was 2 hours, and the yield of Compound E + Compound F was 1.25 mM.

EXAMPLE 6 Synthesis of Compound F, i.e., glyceraldehyde D-3-phosphate, to Compound I, i.e., D-glucose-6-phosphate

The conversion from compound F to compound I can be achieved by three routes, respectively route 11-12-15, route 13-15 and route 14-15 (see FIG. 1 for reaction numbers). First, a catalyst capable of catalyzing each chemical reaction in each pathway was selected (see Table 1), but the enzyme having the catalytic function is not limited to those listed in Table 1. Then combining different catalysts according to a path to establish a corresponding reaction system, and detecting the yield of the compound I after reacting for a period of time.

The production of compound I was measured as follows: 100 μ L of the assay system included Hepes buffer 100mM, NaCl 100mM, 20U/mL 6-phosphoglucose dehydrogenase, 1mM NAD, pH7.5⁺After 20. mu.L of the reaction was terminated, a suitably diluted sample to be tested was taken and the change in OD340 was detected. The yield of compound I was calculated according to a standard curve for glucose-6-phosphate.

Paths 11-12-15: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, Compound F3 mM, Compound E3 mM, FBA 0.1mg/mL, FBP 0.2mg/mL and PGI 0.17 mg/mL; the reaction time was 0.5 hour, and the yield of Compound I was 2.3 mM.

Path 13-15: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, Compound F3 mM, Compound D3 mM, FSA 0.3mg/mL, PGI 0.17 mg/mL; the reaction time was 0.5 hour, and the yield of Compound I was 1.28 mM.

Path 14-15: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, Compound F3 mM, Compound E3 mM, FBAP 0.3mg/mL and PGI 0.17 mg/mL; the reaction time was 0.5 hour, and the yield of Compound I was 0.13 mM.

EXAMPLE 7 Synthesis of Compound I, D-glucose-6-phosphate, to Compound 1, amylose

The conversion from compound I to compound 1 can be achieved by two pathways, pathway 16-17-18 and pathway 16-19, respectively (see figure 1 for reaction numbers). First, a catalyst capable of catalyzing each chemical reaction in each pathway was selected (see Table 1), but the enzyme having the catalytic function is not limited to those listed in Table 1. Then different catalysts are combined according to the path to establish a corresponding reaction system, and after a period of reaction, the yield of the compound 1 is detected.

The production of compound 1 was measured as follows: quantitative determination of Compound 1, after the reaction is terminated, the sample is diluted appropriately, and then incubated with 30U/mL alpha-amylase and 33U/mL glucoamylase for a certain period of time until Compound 1 is completely hydrolyzed to glucose, and then the glucose content is determined by a glucose assay kit (Beijing prilley Gene technology, Inc., E1010). The yield of compound 1 is expressed as glucose content.

Paths 16-17-18: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, Compound I5 mM, ATP 10mM, dextrin 10mg/L, PGM 0.275mg/mL, GlgC 0.46mg/mL, PPase 0.2mg/mL and GlgA 0.235 mg/mL; after 3 hours of reaction, the yield of Compound 1 was 436.8 mg/L.

Paths 16-19: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, Compound I5 mM, dextrin 10mg/L, PGM 0.275mg/mL, and α GP 0.13mg/mL, reacted for 3 hours with compound 1 yield of 100.7 mg/L.

Example 8 Synthesis of Compound 1, amylose, to Compound 2, amylopectin

The conversion from compound 1 to compound 2 can be achieved via pathway 20 (see figure 1 for reaction numbers). First, a catalyst that can catalyze the chemical reaction 20 (see table 1) was selected, but the enzyme having the catalytic function is not limited to those listed in table 1. Then different catalysts are combined according to the path to establish a corresponding reaction system, and after a period of reaction, the yield of the compound 2 is detected.

The production of compound 2 was measured as follows: 1) qualitative detection of compound 2: according to the principle that amylose is changed into blue when meeting iodine solution (the maximum absorption wavelength is about 620 nm), and amylopectin is changed into purple when meeting iodine solution (the maximum absorption wavelength is about 530 nm), whether the compound 2 is generated or not is qualitatively judged by detecting the change of the maximum absorption wavelength of the compound before and after reaction through iodine staining; 2) and (3) quantitatively detecting the compound 2, taking a proper diluted sample after the reaction is terminated, then incubating the diluted sample with 30U/mL alpha-amylase and 33U/mL glucoamylase for a certain time until the compound 2 is completely hydrolyzed into glucose, and then detecting the content of the glucose by using a glucose determination kit (Beijing prilley gene technology Co., Ltd., E1010). The yield of compound 2 is expressed as glucose content.

Path 20: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, compound 13g/L, GlgB 0.05 mg/mL; the reaction time was 3 hours, and the yield of Compound 2 was 2.7 g/L. Fig. 5 shows the full wavelength scan spectra before and after the pathway reaction from compound 1 to compound 2.

Example 9 Synthesis of Compound D, dihydroxyacetone, to Compound 1, amylose

The conversion from compound D to compound I can be achieved by the following pathway:

route 9-10-11-12-15-16-17-18: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, DAK 0.077mg/mL, TPI 0.33mg/mL, FBA 0.15mg/mL, FBP-M0.6 mg/mL, PGI 0.069mg/mL, PGM 0.565mg/mL, GlgA 0.5mg/mL, GlgC-M1 mg/mL, EDTA 0.1mM, ADP 1mM, polyphosphate 0.2mM, PPK 0.22mg/mL (polyphosphate kinase for ATP regeneration), Compound D3 mMDextrin 10 mg/L. After 5 hours of reaction, the yield of Compound 1 was 116.2 mg/L.

Route 9-10-13-15-16-17-18: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, DAK 0.077mg/mL, TPI 0.33mg/mL, FSA 0.3mg/mL, PGI 0.069mg/mL, PGM 0.565mg/mL, GlgA 0.5mg/mL, GlgC-M1 mg/mL, EDTA 0.1mM, ADP 1mM, polyphosphate 0.2mM, PPK 0.22mg/mL (polyphosphate kinase, for ATP regeneration), Compound D3 mM, dextrin 10 mg/L. After 5 hours of reaction, the yield of Compound 1 was 74.5 mg/L.

Route 9-10-14-15-16-17-18: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, DAK 0.077mg/mL, TPI 0.33mg/mL, FBAP 2.5mg/mL, PGI 0.069mg/mL, PGM 0.565mg/mL, GlgA 0.5mg/mL, GlgC-M1 mg/mL, EDTA 0.1mM, ADP 1mM, polyphosphate 0.2mM, PPK 0.22mg/mL (polyphosphate kinase, for ATP regeneration), Compound D3 mM, dextrin 10 mg/L. After 5 hours of reaction, the yield of Compound 1 was 134.4 mg/L.

Route 9-10-13-15-16-19: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, DAK 0.077mg/mL, TPI 0.33mg/mL, FSA 0.3mg/mL, PGI 0.069mg/mL, PGM 0.565mg/mL,. alpha.GP 1mg/mL, EDTA 0.1mM, ADP 1mM, polyphosphate 0.2mM, PPK 0.22mg/mL (polyphosphate kinase, for ATP regeneration), Compound D10mM, dextrin 10 mg/L. After 23 hours of reaction, the yield of Compound 1 was 206.55 mg/L. Figure 6 shows the yield of the pathway from compound D to compound 1.

FBP-M used in example 9 is a fructose-bisphosphatase (FBP) mutant comprising a total of four mutation sites, lysine at position 104 (encoded by AAA) to glutamine (encoded by CAG), arginine at position 132 (encoded by CGC) to isoleucine (encoded by ATT), tyrosine at position 210 (encoded by TAC) to phenylalanine (encoded by TTT), lysine at position 218 (encoded by AAG) to glutamine (encoded by CAG). GlgC-M is a mutant of glucose-1-phosphoadenylyl transferase (GlgC), and contains two mutation sites, namely mutation of proline at position 295 (encoded by CCG) to aspartic acid (encoded by GAT) and mutation of glycine at position 336 (encoded by GGC) to aspartic acid (encoded by GAT). Gene fragments containing the desired mutation were obtained by fusion PCR and cloned into pET21b vector (Novagen, Madison, Wis.) by Simple Cloning (You, C., et al. (2012) 'Simple Cloning via Direct Transformation of PCR products (DNA Multimer) to Escherichia coli and Bacillus subtilis.' Appl. environ. Microbiol.78(5): 1593. 1595.), to obtain the corresponding expression vectors pET21b-FBP-M and pET21 b-GlgC-M. Both plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

EXAMPLE 10 Synthesis of Compound D, dihydroxyacetone, to Compound 2, amylopectin

The synthesis from compound D to compound 2 can be achieved by the following route:

route 9-10-11-12-15-16-17-18-20: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, DAK 0.77mg/mL, TPI 0.33mg/mL, FBA 0.15mg/mL, FBP-M0.43 mg/mL, PGI 0.1mg/mL, PGM 0.565mg/mL, GlgA 0.47mg/mL, GlgC-M0.92 mg/mL, GlgB 0.02mg/mL, EDTA 0.1mM, ADP 1mM, polyphosphate 0.4mM (supplemented with 0.2mM per hour), PPK 0.44mg/mL (polyphosphate kinase for ATP regeneration), Compound D20 mM, dextrin 0.1 g/L. After 4 hours of reaction, the yield of Compound 2 was 1107.77 mg/L. Figure 7 shows the yields of the synthetic pathway from compound D to compound 2.

Example 11 Synthesis of amylose as Compound 1 from methanol

The synthesis of compound 1 from methanol can be achieved by the following route:

route 2-8-9-10-11-12-15-16-17-18: the reaction system is Hepes buffer solution 100mM, NaCl 100mM, Mg of pH7.5²⁺5mM，Zn²⁺10 μ M, AOX 1U/mL, CAT 300U/mL, FLS-M5 mg/mL, thiamine pyrophosphate 0.5mM, DAK 0.035mg/mL, TPI 0.33mg/mL, FBA 0.05mg/mL, FBP-M0.2 mg/mL, PGI 0.023mg/mL, PGM 0.11mg/mL, GlgA 0.1mg/mL, GlgC-M0.2 mg/mL, EDTA 0.1mM, ADP 1mM, polyphosphoric acid 0.2mM, PPK 0.22 mMmg/mL (polyphosphate kinase, for ATP regeneration), methanol 20mM, dextrin 0.01 g/L. After 6 hours of reaction, the yield of Compound 1 was 221.1 mg/L. Figure 8 shows the yield of the synthetic pathway from methanol to compound 1.

FLS-M used in example 11 is a mutant of Formaldehyde Lyase (FLS) and contains three mutation sites, i.e., isoleucine (encoded by ATT) at position 28 to leucine (encoded by CTA), threonine (encoded by ACC) at position 90 to leucine (encoded by CTG), and asparagine (encoded by AAC) at position 283 to histidine (encoded by CAT). A gene fragment containing a mutation of interest is obtained by a fusion PCR method, and cloned into a pET21a vector (Novagen, Madison, Wis.) by a method of Simple Cloning (You, C., et al. (2012) 'Simple Cloning via Direct Transformation of PCR products (DNA Multimer) to Escherichia coli and Bacillus subtilis.' Appl. environ. Microbiol.78(5):1593-1595.), to obtain a corresponding expression vector pET21 a-FLS-M. Both plasmids were transformed into E.coli expression strain BL21(DE3) (Invitrogen, Carlsbad, Calif.) and protein expression and purification were performed.

The FBP-M used in example 11 was the same as that used in example 9.

The above examples illustrate that the process of the invention allows a fully artificial biosynthesis of starch from simple compounds such as dihydroxyacetone, formaldehyde, formic acid and methanol with short cycle times and high yields.

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.

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