STARCH BIOSYNTHESIS METHOD

Abstract

A starch biosynthesis method may implement total artificial biosynthesis from simple compounds such as dihydroxyacetone, formaldehyde, formic acid and methanol to starch. By coupling with methods such as chemical reduction of carbon dioxide, even total artificial biosynthesis of starch taking carbon dioxide as a starting raw material can be implemented. The method can utilize carbon dioxide of high concentration and high density and electric energy and hydrogen energy of high energy density, is more suitable for an industrial production mode, and the production cycle is shortened from several months in farming to several days.

Claims

1-11. (canceled)

12. A method for synthesis of starch, comprising pathway of converting starting material compound D, namely dihydroxyacetone, into starch by catalysis with multiple enzymes; wherein the pathway comprises the following steps: step (1): converting the starting material compound D, namely dihydroxyacetone, into a compound F, namely D-glyceraldehyde 3-phosphate, by catalysis with one or more enzymes; step (2): converting the compound F obtained in step (1) into a compound I, namely D-glucose-6-phosphate, by catalysis with one or more enzymes; and step (3): converting the compound I obtained in step (2) into starch by catalysis with one or more enzymes; the enzyme used in step (1) is an enzyme or a combination of enzymes which catalyzes conversion of dihydroxyacetone into D-glyceraldehyde 3-phosphate by one-step or multi-step reaction; the enzyme used in step (2) is an enzyme or a combination of enzymes which catalyzes conversion of D-glyceraldehyde 3-phosphate into D-glucose-6-phosphate by one-step or multi-step reaction; the enzyme used in step (3) is an enzyme or a combination of enzymes which catalyzes conversion of D-glucose-6-phosphate into amylose or amylopectin by one-step or multi-step reaction.

13. The method for synthesis of starch as claimed in claim 12, wherein step (1) comprises the following sub-steps: step (1-1): converting the compound D into a compound E, namely dihydroxyacetone phosphate, by catalysis with one or more enzymes (the reaction is denoted by reaction 9); and step (1-2): converting the compound E obtained in step (1-1) into the compound F, namely D-glyceraldehyde 3-phosphate, by catalysis with one or more enzymes (the reaction is denoted by reaction 10); the enzyme used in step (1-1) is an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate; the enzyme used in step (1-2) is an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate; wherein the enzyme used in step (1) may be an enzyme combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate and an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate.

14. The method for synthesis of starch as claimed in claim 12, wherein step (2) comprises the following sub-steps: step (2-1): converting the compound F into a compound H, namely D-fructose-6-phosphate, by catalysis with one or more enzymes; the enzyme used in step (2-1) is an enzyme or a combination of enzymes which catalyzes conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate by one-step or multi-step reaction; the enzyme may be a single enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, or may be an enzyme combination (I-2-1): a combination of an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate and an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate; and step (2-2): converting the compound H obtained in step (2-1) into the compound I, namely D-glucose-6-phosphate, by catalysis with one or more enzymes (the reaction is denoted by reaction 15); the enzyme used in step (2-2) is an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate.

15. The method for synthesis of starch as claimed in claim 14, wherein step (2-1) may be done by: converting the compound F into the compound H by catalysis with an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate (the reaction is denoted by reaction 13 or 14); wherein the enzyme used in step (2) may be an enzyme combination (I-2-b): a combination of an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate and an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate.

16. The method for synthesis of starch as claimed in claim 14, wherein step (2-1) may be done by: converting first the compound F into a compound G, namely D-fructose-1,6-bisphosphate, by catalysis with an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate (the reaction is denoted by reaction 11), and then converting the obtained compound G into D-fructose-6-phosphate by catalysis with an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate (the reaction is denoted by reaction 12); wherein the enzyme used in step (2) may be the following enzyme combinations: an enzyme combination (I-2-a): a combination of an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate and an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate.

17. The method for synthesis of starch as claimed in claim 12, wherein step (3) comprises the following sub-steps: step (3-1): converting the compound I into a compound J, namely α-D-glucose-1-phosphate, by catalysis with one or more enzymes (the reaction is denoted by reaction 16); step (3-2): converting the compound J obtained in step (3-1) into a compound 1, namely amylose, by catalysis with one or more enzymes; and optional step (3-3): converting the compound 1 obtained in step (3-2) into a compound 2, namely amylopectin, by catalysis with one or more enzymes (the reaction is denoted by reaction 20); the enzyme used in step (3-1) is an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate; the enzyme used in step (3-2) is an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; the enzyme may be a single enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose, or may be an enzyme combination (I-3-2): a combination of an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; the enzyme used in step (3-3) is an enzyme having the function of catalyzing conversion of amylose into amylopectin.

18. The method for synthesis of starch as claimed in claim 17, wherein step (3-2) may be done by: converting the compound J into the compound 1, namely amylose, by catalysis with an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose (the reaction is denoted by reaction 19); wherein the enzyme used in step (3) may be an enzyme combination (I-3-a): a combination of an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; optionally, the combination (I-3-a) may also comprise an enzyme having the function of catalyzing conversion of amylose into amylopectin.

19. The method for synthesis of starch as claimed in claim 17, wherein step (3-2) may be done by: first, converting the compound J into a compound K, namely adenosine diphosphate-α-D-glucose, by catalysis with an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose (the reaction is denoted by reaction 17), and then converting the obtained compound K into amylose by catalysis with an enzyme having the function of catalyzing adenosine diphosphate-α-D-glucose into amylose (the reaction is denoted by reaction 18); wherein the enzyme used in step (3) may be enzyme combination (I-3-b): a combination of an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; optionally, the combination (I-3-b) may also comprise an enzyme having the function of catalyzing conversion of amylose into amylopectin.

20. The method for synthesis of starch as claimed in claim 12, wherein the method further comprises, before step (1), step (0) of converting starting material formaldehyde into the compound D, namely dihydroxyacetone, by catalysis with one or more enzymes, and the reaction is denoted by reaction 8; the enzyme used in step (0) is an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone.

21. The method for synthesis of starch as claimed in claim 20, wherein the method further comprises, before step (0), step (a) of converting starting material methanol into formaldehyde by catalysis with one or more enzymes; the enzyme used is an enzyme having the function of catalyzing conversion of methanol into formaldehyde.

22. The method for synthesis of starch as claimed in claim 21, comprising the following steps: step 1): converting starting material methanol into amylose by catalysis with multiple enzymes; and optional step 2): converting starting material amylose into amylopectin by catalysis with one or more enzymes; the enzyme used in step 1) is a combination of enzymes which catalyzes synthesis of starch from methanol by multi-step reaction; the enzyme may be the following enzyme combinations: an enzyme combination (II-1-a): a combination of an enzyme having the function of catalyzing conversion of methanol into formaldehyde, an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; an enzyme combination (II-1-b): a combination of an enzyme having the function of catalyzing conversion of methanol into formaldehyde, an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; an enzyme combination (II-1-c): a combination of an enzyme having the function of catalyzing conversion of methanol into formaldehyde, an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; or an enzyme combination (II-1-d): a combination of an enzyme having the function of catalyzing conversion of methanol into formaldehyde, an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; the enzyme used in step 2) is an enzyme having the function of catalyzing conversion of amylose into amylopectin.

23. A method for the synthesis of starch as claimed in claim 21, comprising the following steps: step 1): converting starting material methanol into a compound D, namely dihydroxyacetone, by catalysis with one or more enzymes; step 2): converting the dihydroxyacetone obtained in step 1) into amylose by catalysis with one or more enzymes; and optional step 3): converting the amylose obtained in step 2) into amylopectin by catalysis with one or more enzymes; the enzyme used in step 1) is an enzyme combination (III-1): a combination of an enzyme having the function of catalyzing conversion of methanol into formaldehyde and an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; the enzyme used in step 2) is the following enzyme combinations: an enzyme combination (III-2-a): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; an enzyme combination (III-2-b): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; an enzyme combination (III-2-c): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; or an enzyme combination (III-2-d): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; the enzyme used in step 3) is an enzyme having the function of catalyzing conversion of amylose into amylopectin.

24. The method for synthesis of starch as claimed in claim 20, wherein the method further comprises, before step (0), step (a) of converting starting material formic acid into formaldehyde by catalysis with one or more enzymes; any one of the following steps (a1), (a2) and (a3) can be followed: step (a1): converting starting material formic acid into formaldehyde by catalysis with an enzyme having the function of catalyzing conversion of formic acid into formaldehyde (the reaction is denoted by reaction 3); the enzyme used in step(a) may be a single enzyme having the function of catalyzing conversion of formic acid into formaldehyde; step (a2): first, converting starting material formic acid into formyl coenzyme A by catalysis with an enzyme having the function of catalyzing conversion of formic acid into formyl coenzyme A (the reaction is denoted by reaction 4), and then converting formyl coenzyme A into formaldehyde by catalysis with an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde (the reaction is denoted by reaction 7); the enzyme used in step(a) may be an enzyme combination (I-a-1): a combination of an enzyme having the function of catalyzing conversion of formic acid into formyl coenzyme A and an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde; or step (a3): first, converting starting material formic acid into formyl phosphate by catalysis with an enzyme having the function of catalyzing conversion of formic acid into formyl phosphate (the reaction is denoted by reaction 5), then converting formyl phosphate into formyl coenzyme A by catalysis with an enzyme having the function of catalyzing conversion of formyl phosphate into formyl coenzyme A (the reaction is denoted by reaction 6), and then converting formyl coenzyme A into formaldehyde by catalysis with an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde (the reaction is denoted by reaction 7); the enzyme used in step(a) may be an enzyme combination (I-a-2): a combination of an enzyme having the function of catalyzing conversion of formic acid into formyl phosphate, an enzyme having the function of catalyzing conversion of formyl phosphate into formyl coenzyme A, and an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde.

25. A method for the synthesis of starch as claimed in claim 24, comprising the following steps: step 1): converting starting material formic acid into dihydroxyacetone by catalysis with one or more enzymes; step 2): converting the dihydroxyacetone obtained in step 1) into amylose by catalysis with one or more enzymes; and optional step 3): converting the amylose obtained in step 2) into amylopectin by catalysis with one or more enzymes; the enzyme used in step 1) is an enzyme or a combination of enzymes which catalyzes conversion of formic acid into dihydroxyacetone by one-step or multi-step reaction; the enzyme may be the following enzyme combinations: an enzyme combination (IV-1-a): a combination of an enzyme having the function of catalyzing conversion of formic acid into formaldehyde and an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; an enzyme combination (IV-1-b): a combination of an enzyme having the function of catalyzing conversion of formic acid into formyl coenzyme A, an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde and an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; or an enzyme combination (IV-1-c): a combination of an enzyme having the function of catalyzing conversion of formic acid into formyl phosphate, an enzyme having the function of catalyzing conversion of formyl phosphate into formyl coenzyme A, an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde and and an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; the enzyme used in step 2) is an enzyme or a combination of enzymes which catalyzes conversion of dihydroxyacetone into amylose by one-step or multi-step reaction; the enzyme may be following enzyme combinations: an enzyme combination (IV-2-a): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; an enzyme combination (IV-2-b): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; an enzyme combination (IV-2-c): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; or an enzyme combination (IV-2-d): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; the enzyme used in step 3) is an enzyme having the function of catalyzing conversion of amylose into amylopectin.

26. A method for the synthesis of starch as claimed in claim 14, comprising the following steps: step 1): converting starting material formic acid into formaldehyde by catalysis with one or more enzymes; step 2): converting the formaldehyde obtained in step 1) into dihydroxyacetone by catalysis with one or more enzymes; step 3): converting the dihydroxyacetone obtained in step 2) into amylose by catalysis with one or more enzymes; and optional step 4): converting the amylose obtained in step 3) into amylopectin by catalysis with one or more enzymes; the enzyme used in step 1) is a single enzyme having the function of catalyzing conversion of formic acid into formaldehyde, or the following enzyme combinations: an enzyme combination (V-1-a): an enzyme having the function of catalyzing conversion of formic acid into formyl coenzyme A and an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde, or an enzyme combination (V-1-b): a combination of an enzyme having the function of catalyzing conversion of formic acid into formyl phosphate, an enzyme having the function of catalyzing conversion of formyl phosphate into formyl coenzyme A and an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde; the enzyme used in step 2) is an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; the enzyme used in step 3) is the following enzyme combinations: an enzyme combination (V-3-a): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; an enzyme combination (V-3-b): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; an enzyme combination (V-3-c): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; or an enzyme combination (V-3-d): a combination of an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate, an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate, an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate, an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate, an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; the enzyme used in step 4) is an enzyme having the function of catalyzing conversion of amylose into amylopectin.

27. A method for the synthesis of starch as claimed in claim 24, comprising the following steps: step 1): converting starting material formic acid into formaldehyde by catalysis with one or more enzymes; step 2): converting the formaldehyde obtained in step 1) into a compound F, namely D-glyceraldehyde 3-phosphate, by catalysis with one or more enzymes; step 3): converting the D-glyceraldehyde 3-phosphate obtained in step 2) into D-glucose-6-phosphate by catalysis with one or more enzymes; step 4): converting the D-glucose-6-phosphate obtained in step 3) into amylose by catalysis with one or more enzymes; and optional step 5): converting the amylose obtained in step 4) into amylopectin by catalysis with one or more enzymes; the enzyme used in step 1) is an enzyme combination (VI-1): a combination of an enzyme having the function of catalyzing conversion of formic acid into formyl phosphate, an enzyme having the function of catalyzing conversion of formyl phosphate into formyl coenzyme A and an enzyme having the function of catalyzing conversion of formyl coenzyme A into formaldehyde; the enzyme used in step 2) is an enzyme combination (VI-2): a combination of an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone, an enzyme having the function of catalyzing conversion of dihydroxyacetone into dihydroxyacetone phosphate and an enzyme having the function of catalyzing conversion of dihydroxyacetone phosphate into D-glyceraldehyde 3-phosphate; the enzyme used in step 3) is the following enzyme combinations: an enzyme combination (VI-3-a): a combination of an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-1,6-bisphosphate, an enzyme having the function of catalyzing conversion of D-fructose-1,6-bisphosphate into D-fructose-6-phosphate and an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate; or an enzyme combination (VI-3-b): a combination of an enzyme having the function of catalyzing conversion of D-glyceraldehyde 3-phosphate into D-fructose-6-phosphate and an enzyme having the function of catalyzing conversion of D-fructose-6-phosphate into D-glucose-6-phosphate; the enzyme used in step 4) is the following enzyme combinations: an enzyme combination (VI-4-a): a combination of an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate, and enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into adenosine diphosphate-α-D-glucose and an enzyme having the function of catalyzing conversion of adenosine diphosphate-α-D-glucose into amylose; or an enzyme combination (VI-4-b): a combination of an enzyme having the function of catalyzing conversion of D-glucose-6-phosphate into α-D-glucose-1-phosphate and an enzyme having the function of catalyzing conversion of α-D-glucose-1-phosphate into amylose; the enzyme used in step 5) is an enzyme having the function of catalyzing conversion of amylose into amylopectin.

28. The method for synthesis of starch as claimed in any one of claims 12, wherein the steps, sub-steps or specific reactions of the method can be performed step by step, or any adjacent two, three, four, five, six, seven or more steps, sub-steps or specific reactions can also be performed simultaneously, or all the steps or specific reactions can be performed simultaneously.

29. A method for the synthesis of dihydroxyacetone, comprising pathway of converting starting material methanol into dihydroxyacetone by catalysis with one or more enzymes; wherein the pathway comprises the following steps: step (1): converting starting material methanol into formaldehyde by catalysis with an enzyme having the function of catalyzing conversion of methanol into formaldehyde; and step (2): converting the formaldehyde obtained in step (1) into dihydroxyacetone by catalysis with an enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; preferably, the enzyme having the function of catalyzing conversion of methanol into formaldehyde in step (1) includes, but is not limited to, alcohol oxidase (AOX) or mutants thereof, cholesterol oxidase or mutants thereof, alcohol dehydrogenase (ADH) or mutants thereof, methanol dehydrogenase or mutants thereof, L-threonine-3-dehydrogenase or mutants thereof, cyclohexanol dehydrogenase or mutants thereof, or n-butanol dehydrogenase or mutants thereof; the enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone in step (2) includes, but is not limited to, formolase (FLS) or mutants thereof (FLS-M), or glycolaldehyde synthase (GALS) or mutants thereof; wherein steps (1) and (2) can be performed simultaneously or step by step; when steps (1) and (2) are simultaneously performed, a reaction system comprises substrate methanol, the enzyme having the function of catalyzing conversion of methanol into formaldehyde and the enzyme having the function of catalyzing conversion of formaldehyde into dihydroxyacetone; optionally, an auxiliary enzyme such as catalase may also be comprised.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0152] FIG. 1 shows a synthesis pathway for the synthesis of compound 1, namely amylose, and compound 2, namely amylopectin, from starting material methanol or formic acid.

[0153] FIG. 2 shows a schematic of the structure of compound 1.

[0154] FIG. 3 shows a schematic of the structure of compound 2.

[0155] FIG. 4 shows the yield of the synthesis pathway from methanol to compound D (i.e. dihydroxyacetone).

[0156] FIG. 5 shows full-wavelength scanning spectra before and after the reaction of the pathway from compound 1 (i.e. amylose) to compound 2 (i.e. amylopectin).

[0157] FIG. 6 shows the yields of different pathways from compound D to compound 1.

[0158] FIG. 7 shows the yield of a different pathway from compound D to compound 2.

[0159] FIG. 8 shows the yield of the synthesis pathway from methanol to compound 1.

DETAILED DESCRIPTION

[0160] The synthesis method disclosed herein will be further illustrated in detail with reference to the following specific examples. It should be understood that the following embodiments are merely exemplary illustration and explanation of the present disclosure, and should not be construed as limiting the protection scope of the present disclosure. All techniques implemented based on the content of the present disclosure described above are encompassed within the protection scope of the present disclosure.

[0161] Unless otherwise stated, the starting materials and reagents used in the following examples are all commercially available products or can be prepared using known methods.

[0162] The catalysts (enzymes) used for the reactions involved in the examples are shown in Table 1 below unless otherwise stated:

TABLE-US-00001 TABLE 1 The catalysts used for the reactions involved in the examples Origin/Gene Reaction Full name of catalyst Abbreviation Host information 1 Alcohol dehydrogenase ADH Bacillus Sigma (A7011) methanolicus 2 Alcohol oxidase AOX Pichia pastoris Sigma (A2404) 3 Aldehyde FADH Burkholderia BAG46824.1 dehydrogenase multivorans 4 Acetyl-coenzyme A ACS Escherichia coli AAC77039.1 synthetase 5 Acetate kinase ACKA Escherichia coli AAC75356.1 6 Phosphate PTA Escherichia coli AAC75511.1 acetyltransferase 7 Acetaldehyde ACDH Listeria CAC99712.1 dehydrogenase monocytogenes 8 Formolase FLS Pseudomonas # putida 9 Dihydroxyacetone DAK Pichia pastoris AAC39490.1 kinase 10 Triose phosphate TPI Escherichia coli AAC76901.1 isomerase 11 Fructose-bisphosphate FBA Escherichia coli AAC75158.2 aldolase 12 Fructose-bisphosphatase FBP Escherichia coli AAC77189.1 13 Fructose 6-phosphate FSA Escherichia coli AAC73912.2 aldolase 14 Fructose 6-phosphate FBAP Cenarchaeum ABK77197.1 aldolase phosphatase symbiosum 15 Glucose phosphate PGI Escherichia coli AAC76995.1 isomerase 16 Phosphohexose PGM Lactococcus CAL97144.1 phosphate mutase Lactis 17 Glucose-1-phosphate GlgC Escherichia coli AAC76455.1 adenylyltransferase 18 Starch synthase GlgA Escherichia coli AAC76454.1 19 Starch phosphorylase αGP Hordeum vulgare BAK00834.1 20 1,4-α-D-Glucan GlgB Vibrio vulnificus ADV88080.1 branching enzyme Auxiliary Catalase CAT Bacillus subtilis CAB12710.2 enzyme 168 Formate dehydrogenase FDH Thiobacillus sp. BAC92737.1 KNK65MA Inorganic PPase Escherichia coli AAC77183.1 pyrophosphatase #: FLS gene information is available in the document (Siegel, J.B., et al., Computational protein design enables a novel one-carbon assimilation pathway. Proc Natl Acad Sci USA, 2015. 112(12): p.3704-9.).

[0163] Alcohol dehydrogenase and alcohol oxidase were purchased from Sigma (https://www.sigmaaldrich.com/china-mainland.html). Aldehyde dehydrogenase, acetyl-formyl coenzyme A synthetase, acetate kinase, phosphate acetyltransferase, acetaldehyde dehydrogenase, catalase, formate dehydrogenase and inorganic pyrophosphatase were obtained by PCR or gene synthesis and were cloned into vectors pET20b, pET21b, pET26b and pET28a (Novagen, Madison, WI) by simple cloning (You, C., et al. (2012). “Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis.” Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vectors pET28a-FADH, pET21b-ACS, pET28a-ACKA, pET28a-PTA, pET21b-ACDH, pET26b-CAT, pET20b-FDH and pET21b-PPase. These eight plasmids were all transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

[0164] Formolase, dihydroxyacetone kinase and triose phosphate isomerase were obtained by PCR or gene synthesis and were cloned into pET21a, pET21b and pET28a vectors (Novagen, Madison, WI) respectively by simple cloning (You, C., et al. (2012). Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vectors pET21a-FLS, pET21b-TPI and pET28a-DAK. These three plasmids were all transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

[0165] Fructose-bisphosphate aldolase, fructose-bisphosphatase, fructose 6-phosphate aldolase, fructose 6-phosphate aldolase phosphatase and glucose phosphate isomerase were obtained by PCR or gene synthesis and were cloned into pET21b vector (Novagen, Madison, WI) by simple cloning (You, C., et al. (2012). Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vectors pET21b-FBA, pET21b-FBP, pET21b-FSA, pET21b-FBAP and pET21b-PGI. These five plasmids were all transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

[0166] Phosphohexose phosphate mutase, glucose-1-phosphate adenylyltransferase, starch synthase and starch phosphorylase were obtained by PCR or gene synthesis and were cloned into pET20b and pET21b vectors (Novagen, Madison, WI) by simple cloning (You, C., et al. (2012). Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vectors pET21b-PGM, pET21b-GlgC, pET21b-GlgA and pET20b-αGP. These four plasmids were all transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

[0167] 1,4-α-D-Glucan branching enzyme was obtained by PCR or gene synthesis and was cloned into pET28a vectors (Novagen, Madison, WI) by simple cloning (You, C., et al. (2012). Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vector pET28a-GlgB. This plasmid was transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the protein was expressed and purified.

Example 1. Synthesis of Compound C, Namely Formaldehyde, from Formic Acid or Methanol

[0168] There were five pathways to achieve the conversion of formic acid or methanol into compound C: Pathway 1 (from methanol to formaldehyde), Pathway 2 (from methanol to formaldehyde), Pathway 3 (from formic acid to formaldehyde), Pathway 4-7 (from formic acid to formyl coenzyme A to formaldehyde) and Pathway 5-6-7 (from formic acid to formyl phosphate, then to formyl coenzyme A, and then to formaldehyde) (the reactions were numbered as shown in FIG. 1). First, the catalysts (i.e., enzymes) capable of catalyzing the chemical reactions of the pathways were selected (see Table 1); however, enzymes having the corresponding catalytic functions are not limited to those listed in Table 1. Then different catalysts were combined according to the pathways, and corresponding reaction systems were established. After the reactions were performed for a period of time, the yield of formaldehyde was determined.

[0169] The yield of compound C was determined as follows: to 200 μL of water was added 50 μL of test solution (was properly diluted)and then 25 μL of acetylacetone solution (100 mL of the acetylacetone solution comprised 0.5 mL of acetylacetone, 50 g of ammonium acetate and 6 mL of glacial acetic acid); the mixture was reacted at 60° C. for 15 min and then centrifuged; 200 μL of the supernatant was collected and assayed for the OD414 value, and the compound C content was calculated from the formaldehyde standard curve.

[0170] Pathway 1: The reaction system comprised 100 mM Tris buffer with pH of 8.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 3.5 mg/mL ADH, 100 mM NAD.sup.+ and 1 M methanol. The reaction was performed for 3 h, and the yield of formaldehyde was 0.27 mM.

[0171] Pathway 2: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 1 U/mL AOX, 300 U/mL CAT (auxiliary enzyme for eliminating hydrogen peroxide generated by AOX) and 20 mM methanol. The reaction was performed for 0.5 h, and the yield of formaldehyde was 12 mM.

[0172] Pathway 3: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 4 mg/mL FADH, 100 mM NADH and 250 mM sodium formate. The reaction was performed for 3 h, and the yield of formaldehyde was 0.1 mM.

[0173] Pathway 4-7: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 2 mM ATP, 1.5 mM NADH, 0.1 mM CoA, 3.7 mg/mL ACS, 0.2 mg/mL ACDH, 0.024 mg/mL FDH (auxiliary enzyme for regenerating NADH), 0.1 mg/mL PPase (auxiliary enzyme for hydrolyzing pyrophosphoric acid) and 50 mM sodium formate. The reaction was performed for 1 h, and the yield of formaldehyde was 0.6 mM.

[0174] Pathway 5-6-7: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 0.5 mM NADH, 10 mM ATP, 0.1 mM CoA, 2 mM β-mercaptoethanol, 0.24 mg/mL ACKA, 1.2 mg/mL PTA, 0.2 mg/mL ACDH, 0.024 mg/mL FDH (auxiliary enzyme for regenerating NADH) and 50 mM sodium formate. The reaction was performed for 1 h, and the yield of formaldehyde was 2 mM.

Example 2. Synthesis of Compound D, Namely Dihydroxyacetone, from Methanol

[0175] The synthesis of compound D from methanol can be achieved via the pathway below.

[0176] Pathway 2-8: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 1 U/mL AOX, 300 U/mL CAT, 20 mM methanol and 5 mg/mL FLS. The reaction was performed for 2 h, and the yield of formaldehyde was 2.1 mM.

[0177] Assay method for compound D: 5 mM dilute sulfuric acid was used as the mobile phase; an HX87 column (Bio-Rad, Aminex@, 300 mm×78 mm) was used; the flow rate was 0.6 mL/min; the injection amount was 10 μL/injection. The yield of compound D was calculated from the DHA standard curve. the yield of the pathway 2-8 from methanol to compound D was shown in FIG. 4.

Example 3. Synthesis of Compound D, Namely Dihydroxyacetone, from Formaldehyde

[0178] The synthesis of compound D from formaldehyde can be achieved via the pathway below.

[0179] Pathway 8: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 25 mM formaldehyde, 0.5 mM TPP and 10 mg/mL FLS (derived from Pseudomonas putida). The reaction was performed for 2 h. Compound D was detected using the method in Example 2, and the result showed that the yield of compound D synthesized using formolase (FLS) was 7.092 mM.

Example 4. Synthesis of Compound D, Namely Dihydroxyacetone, from Formic Acid

[0180] The synthesis of compound D from formic acid can be achieved via the pathway below.

[0181] Pathway 5-6-7-8: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 50 mM sodium formate, 0.5 mM TPP, 0.1 mM CoA, 10 mM ATP, 2 mM β-mercaptoethanol, 0.24 mg/mL ACKA, 1.2 mg/mL PTA, 0.2 mg/mL ACDH, 0.024 mg/mL FDH and 0.5 mM NADH. After the reaction was performed at 30° C. for 1-1.5 h, 7.5 mg/mL FLS (derived from Pseudomonas putida) was added. After the reaction was performed at 30° C. for another 3.5 h, compound D was detected by gas chromatography-mass spectrometry, and the result showed that the yield of compound D synthesized via the pathway 5-6-7-8 was 0.23 mM.

[0182] The gas chromatography-mass spectrometry detection of compound D was performed as follows: [0183] 1) Sample preparation: 500 μL of properly diluted sample was reacted with 100 μL of 200 mM PFBOA (O-(2,3,4,5,6-Pentafluorobenzyl) hydroxylamine hydrochloride) at 30° C. for 1 h. 100 μL of n-hexane was added for extraction. 40 μL of the organic phase was added to an equal volume of MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide), and the mixture was reacted at 37° C. for 2 h. [0184] 2) Gas chromatography conditions: Agilent 7890A gas chromatograph, helium as carrier gas, carrier gas flow rate of 1.2 mL/min, DB-5MS Ultra Inert capillary column (30 m×250 μm×0.25 μm) as chromatography column, initial temperature of 60° C., maintained for 1 min, running for 1 min, warming rate 1, 5° C./min to 240° C., running for 9 min, warming rate 2, 25° C./min to 300° C., maintained for 5 min, running for 22 min, injection amount of 1 μL/injection, injector temperature of 250° C. [0185] 3) Mass spectrometry conditions: Agilent 7200 Q-TOF mass spectrometer, solvent delay set to 4 min, EI ionization mode, electron energy of 70 eV, ion source temperature: 230° C., scan range: 35-550 amu, mass spectrometry acquisition rate of 5 spectra/s. [0186] 4) Data analysis: The target metabolites were determined using the Mass Hunter software

[0187] Qualitative Analysis by making comparisons to the NIST chemical database and by manual analysis, and the yield of DHA was calculated.

Example 5. Synthesis of Compound F, Namely D-Glyceraldehyde 3-Phosphate, from Compound C, Namely Formaldehyde

[0188] The conversion of compound C into compound F can be achieved via Pathway 8-9-10 (the reactions were numbered as shown in FIG. 1). First, the catalysts capable of catalyzing the chemical reactions of the pathway were selected (see Table 1); however, enzymes having the catalytic functions are not limited to those listed in Table 1. Then different catalysts were combined according to the pathway, and corresponding reaction system was established. After the reactions were performed for a period of time, the yield of compound F was determined.

[0189] Compound E and compound F are isomers, but the cumulative amount of compound F would be low due to the unfavorable free energy, so the yield of compound E+compound F was determined. The yield of compound E+compound F was determined as follows: a 100 μL assay system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 20 U/mL glyceraldehyde phosphate dehydrogenase, 20 U/mL triose phosphate isomerase, 1 mM NAD.sup.+ and 4 mM potassium arsenate; after the reaction was terminated, a proper dilution of the test sample was assayed for the change in OD340. The yield of compound E+compound F was calculated from the standard curve for glyceraldehyde 3-phosphate.

[0190] Pathway 8-9-10: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 10 mM ATP, 0.5 mM thiamine pyrophosphate, 5 mg/mL FLS, 0.1 mg/mL DAK, 0.14 mg/mL TPI and 10 mM compound C. The reaction was performed for 2 h, and the yield of compound E+compound F was 1.25 mM.

Example 6. Synthesis of Compound I, Namely D-Glucose-6-Phosphate, from Compound F, Namely D-Glyceraldehyde 3-Phosphate

[0191] The conversion of compound F into compound I can be achieved via three pathways: Pathway 11-12-15, Pathway 13-15 and Pathway 14-15 (the reactions were numbered as shown in FIG. 1). First, the catalysts capable of catalyzing the chemical reactions of the pathways (see Table 1) were selected; however, enzymes having the catalytic functions are not limited to those listed in Table 1. Then different catalysts were combined according to the pathways, and corresponding reaction systems were established. After the reactions were performed for a period of time, the yield of compound I was determined.

[0192] The yield of compound I was determined as follows: a 100 μL assay system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 20 U/mL glucose 6-phosphate dehydrogenase and 1 mM NAD.sup.+; after 20 μL of the reaction was terminated, a proper dilution of the test sample was assayed for the change in OD340. The yield of compound I was calculated from the standard curve for glucose 6-phosphate. Pathway 11-12-15: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 3 mM compound F, 3 mM compound E, 0.1 mg/mL FBA, 0.2 mg/mL FBP and 0.17 mg/mL PGI. The reaction was performed for 0.5 h, and the yield of compound I was 2.3 mM.

[0193] Pathway 13-15: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 3 mM compound F, 3 mM compound D, 0.3 mg/mL FSA and 0.17 mg/mL PGI. The reaction was performed for 0.5 h, and the yield of compound I was 1.28 mM.

[0194] Pathway 14-15: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 3 mM compound F, 3 mM compound E, 0.3 mg/mL FBAP and 0.17 mg/mL PGI. The reaction was performed for 0.5 h, and the yield of compound I was 0.13 mM.

Example 7. Synthesis of Compound 1, Namely Amylose, from Compound I, Namely D-Glucose-6-Phosphate

[0195] The conversion of compound I into compound 1 can be achieved via two pathways: Pathway 16-17-18 and Pathway 16-19 (the reactions were numbered as shown in FIG. 1). First, the catalysts capable of catalyzing the chemical reactions of the pathways (see Table 1) were selected; however, enzymes having the catalytic functions are not limited to those listed in Table 1. Then different catalysts were combined according to the pathways, and corresponding reaction systems were established. After the reactions were performed for a period of time, the yield of compound 1 was determined.

[0196] The yield of compound 1 was determined as follows: after the reaction was terminated, the sample was properly diluted, and then incubated with 30 U/mL α-amylase and 33 U/mL glucoamylase for a certain period of time until compound 1 was completely hydrolyzed to glucose, and then the glucose content was determined using a glucose assay kit (Beijing Applygen Technologies Inc., E1010). The yield of compound 1 is expressed in terms of the glucose content.

[0197] Pathway 16-17-18: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 5 mM compound I, 10 mM ATP, 10 mg/L dextrin, 0.275 mg/mL PGM, 0.46 mg/mL GlgC, 0.2 mg/mL PPase and 0.235 mg/mL GlgA. The reaction was performed for 3 h, and the yield of compound 1 was 436.8 mg/L.

[0198] Pathway 16-19: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 5 mM compound I, 10 mg/L dextrin, 0.275 mg/mL PGM and 0.13 mg/mL αGP. The reaction was performed for 3 h, and the yield of compound 1 was 100.7 mg/L.

Example 8. Synthesis of Compound 2, Namely Amylopectin, from Compound 1, Namely Amylose

[0199] The conversion of compound 1 into compound 2 can be achieved via Pathway 20 (the reactions were numbered as shown in FIG. 1). First, the catalyst capable of catalyzing the chemical reaction 20 (see Table 1) was selected; however, an enzyme having the catalytic function is not limited to that listed in Table 1. Then different catalysts were combined according to the pathways, and corresponding reaction systems were established. After the reactions were performed for a period of time, the yield of compound 2 was determined.

[0200] The yield of compound 2 was determined as follows: 1) qualitative detection of compound 2: based on the principle that amylose turns blue in the presence of iodine solution (the maximum absorption wavelength is about 620 nm), and that amylopectin turns purple in the presence of iodine solution (the maximum absorption wavelength is about 530 nm), whether compound 2 was produced was qualitatively determined by detecting the change in the maximum absorption wavelength of the compound before and after reaction by iodine staining; 2) quantitative measurement of compound 2: after the reaction was terminated, a proper dilution of the sample was incubated with 30 U/mL α-amylase and 33 U/mL glucoamylase for a certain period of time until compound 2 was completely hydrolyzed to glucose, and then the glucose content was determined using a glucose assay kit (Beijing Applygen Technologies Inc., E1010). The yield of compound 2 is expressed in terms of the glucose content.

[0201] Pathway 20: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 3 g/L compound 1 and 0.05 mg/mL GlgB. The reaction was performed for 3 h, and the yield of compound 2 was 2.7 g/L. FIG. 5 shows full-wavelength scanning spectra before and after the reaction of the pathway from compound 1 to compound 2.

Example 9. Synthesis of Compound 1, Namely Amylose, from Compound D, Namely Dihydroxyacetone

[0202] The conversion of compound D into compound I can be achieved via the pathway below.

[0203] Pathway 9-10-11-12-15-16-17-18: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 0.077 mg/mL DAK, 0.33 mg/mL TPI, 0.15 mg/mL FBA, 0.6 mg/mL FBP-M, 0.069 mg/mL PGI, 0.565 mg/mL PGM, 0.5 mg/mL GlgA, 1 mg/mL GlgC-M, 0.1 mM EDTA, 1 mM ADP, 0.2 mM polyphosphoric acid, 0.22 mg/mL PPK (polyphosphate kinase, for regenerating ATP), 3 mM compound D and 10 mg/L dextrin. The reaction was performed for 5 h, and the yield of compound 1 was 116.2 mg/L.

[0204] Pathway 9-10-13-15-16-17-18: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 0.077 mg/mL DAK, 0.33 mg/mL TPI, 0.3 mg/mL FSA, 0.069 mg/mL PGI, 0.565 mg/mL PGM, 0.5 mg/mL GlgA, 1 mg/mL GlgC-M, 0.1 mM EDTA, 1 mM ADP, 0.2 mM polyphosphoric acid, 0.22 mg/mL PPK (polyphosphate kinase, for regenerating ATP), 3 mM compound D and 10 mg/L dextrin. The reaction was performed for 5 h, and the yield of compound 1 was 74.5 mg/L.

[0205] Pathway 9-10-14-15-16-17-18: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 0.077 mg/mL DAK, 0.33 mg/mL TPI, 2.5 mg/mL FBAP, 0.069 mg/mL PGI, 0.565 mg/mL PGM, 0.5 mg/mL GlgA, 1 mg/mL GlgC-M, 0.1 mM EDTA, 1 mM ADP, 0.2 mM polyphosphoric acid, 0.22 mg/mL PPK (polyphosphate kinase, for regenerating ATP), 3 mM compound D and 10 mg/L dextrin. The reaction was performed for 5 h, and the yield of compound 1 was 134.4 mg/L.

[0206] Pathway 9-10-13-15-16-19: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 0.077 mg/mL DAK, 0.33 mg/mL TPI, 0.3 mg/mL FSA, 0.069 mg/mL PGI, 0.565 mg/mL PGM, 1 mg/mL αGP, 0.1 mM EDTA, 1 mM ADP, 0.2 mM polyphosphoric acid, 0.22 mg/mL PPK (polyphosphate kinase, for regenerating ATP), 10 mM compound D and 10 mg/L dextrin. The reaction was performed for 23 h, and the yield of compound 1 was 206.55 mg/L. FIG. 6 shows the yields of the pathways from compound D to compound 1. FBP-M used in Example 9 is a mutant of fructose-bisphosphatase (FBP), which comprises a total of four mutated sites: lysine (encoded by AAA) at position 104 mutated to glutamine (encoded by CAG), arginine (encoded by CGC) at position 132 mutated to isoleucine (encoded by ATT), tyrosine (encoded by TAC) at position 210 mutated to phenylalanine (encoded by TTT), and lysine (encoded by AAG) at position 218 mutated to glutamine (encoded by CAG). GlgC-M is a mutant of glucose-1-phosphate adenylyltransferase (GlgC), which comprises a total of two mutated sites: proline (encoded by CCG) at position 295 mutated to aspartic acid (encoded by GAT), and glycine (encoded by GGC) at position 336 mutated to aspartic acid (encoded by GAT). Gene fragments comprising the desired mutations were obtained by fusion PCR and were all cloned into pET21b vector (Novagen, Madison, WI) by simple cloning (You, C., et al. (2012). “Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis.” Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vectors pET21b-FBP-M and pET21b-GlgC-M. These two plasmids were transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

Example 10. Synthesis of Compound 2, Namely Amylopectin, from Compound D, Namely Dihydroxyacetone

[0207] The synthesis of compound 2 from compound D can be achieved via the pathway below.

[0208] Pathway 9-10-11-12-15-16-17-18-20: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 0.77 mg/mL DAK, 0.33 mg/mL TPI, 0.15 mg/mL FBA, 0.43 mg/mL FBP-M, 0.1 mg/mL PGI, 0.565 mg/mL PGM, 0.47 mg/mL GlgA, 0.92 mg/mL GlgC-M, 0.02 mg/mL GlgB, 0.1 mM EDTA, 1 mM ADP, 0.4 mM polyphosphoric acid (additional 0.2 mM was added per hour), 0.44 mg/mL PPK (polyphosphate kinase, for regenerating ATP), 20 mM compound D and 0.1 g/L dextrin. The reaction was performed for 4 h, and the yield of compound 2 was 1107.77 mg/L. FIG. 7 shows the yield of the pathway from compound D to compound 2.

Example 11. Synthesis of Compound 1, Namely Amylose, from Methanol

[0209] The synthesis of compound 1 from methanol can be achieved via the pathway below.

[0210] Pathway 2-8-9-10-11-12-15-16-17-18: The reaction system comprised 100 mM Hepes buffer with pH of 7.5, 100 mM NaCl, 5 mM Mg.sup.2+, 10 μM Zn.sup.2+, 1 U/mL AOX, 300 U/mL CAT, 5 mg/mL FLS-M, 0.5 mM thiamine pyrophosphate, 0.035 mg/mL DAK, 0.33 mg/mL TPI, 0.05 mg/mL FBA, 0.2 mg/mL FBP-M, 0.023 mg/mL PGI, 0.11 mg/mL PGM, 0.1 mg/mL GlgA, 0.2 mg/mL GlgC-M, 0.1 mM EDTA, 1 mM ADP, 0.2 mM polyphosphoric acid, 0.22 mg/mL PPK (polyphosphate kinase, for regenerating ATP), 20 mM methanol and 0.01 g/L dextrin. The reaction was performed for 6 h, and the yield of compound 1 was 221.1 mg/L. FIG. 8 shows the yield of the synthesis pathway from methanol to compound 1.

[0211] FLS-M used in Example 11 is a mutant of formolase (FLS), which comprises a total of three mutated sites: isoleucine (encoded by ATT) at position 28 mutated to leucine (encoded by CTA), threonine (encoded by ACC) at position 90 mutated to leucine (encoded by CTG), and asparagine (encoded by AAC) at position 283 mutated to histidine (encoded by CAT). Gene fragments comprising the desired mutations were obtained by fusion PCR and were cloned into pET21a vector (Novagen, Madison, WI) by simple cloning (You, C., et al. (2012). “Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis.” Appl. Environ. Microbiol. 78(5):1593-1595.) to obtain corresponding expression vector pET21a-FLS-M. These two plasmids were transformed into Escherichia coli expression system BL21(DE3) (Invitrogen, Carlsbad, CA), and the proteins were expressed and purified.

[0212] FBP-M used in Example 11 was the same as that used in Example 9.

[0213] The above examples show that the total artificial biosynthesis of starch from simple compounds such as dihydroxyacetone, formaldehyde, formic acid and methanol and the like can be achieved by using the methods of the present disclosure and with short periods and high yields.

[0214] The embodiments of the present disclosure have been described above. However, the present disclosure is not limited to the embodiments described above. Any modification, equivalent, improvement and the like made without departing from the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure.