FIELD

The present invention relates to a novel method for producing 4-aminocinnamic acid, which is useful as a raw material monomer for biomass-derived aromatic polymers, as well as to a novel vector and host cell for use in the method.

BACKGROUND

Concerns over exhaustion of petroleum resources and the issue of carbon dioxide emissions have increased the importance of systems for producing fuel and chemical products using biomass, which is a renewable resource. While examples of biomass-derived polymers include aliphatic polymers and aromatic polymers, research and development are currently progressing mainly on aliphatic polymers such as polylactic acid. Polylactic acid has been challenged by low heat resistance and durability, but this problem is being solved by, e.g., improvement in crystallinity. On the other hand, aromatic polymers often exhibit excellent material properties in terms of thermal stability and mechanical strength, and are expected to be used as raw materials for engineering plastics.

Of particular interest as a raw material monomer for biomass-derived aromatic polymers is 4-aminocinnamic acid. Patent Document 1 and Non-Patent Document 1, for example, report a method for synthesizing an aromatic polymer excellent in high heat resistance using 4-aminocinnamic acid as a raw material. Accordingly, there is a demand for a method of synthesizing 4-aminocinnamic acid from biomass with high efficiency, as a raw material monomer of such a high heat-resistance polymer.

The present inventors have developed a method for synthesizing 4-aminocinnamic acid from glucose in biomass using microbial-derived enzymes along a route wherein glucose is first converted to chorismic acid, then to 4-aminophenylpyruvate, and finally to 4-aminophenylalanine (Patent Document 2), which 4-aminophenylalanine is then converted to 4-aminocinnamic acid (Patent Document 3).



CITATION LIST

Patent Documents

[Patent Document 1] WO2013/073519A

[Patent Document 2] WO2015/141791A

[Patent Document 3] WO2015/119251A

NON-PATENT DOCUMENTS

[Non-Patent Document 1] Suvannasara et al., Macromolecules, (2014), 47[5]1586−1593

SUMMARY

Technical Problem

Although the present inventors' method for synthesizing 4-aminocinnamic acid mentioned above is an excellent method, there is still room for improvement in terms of reaction rate and reaction efficiency.

An objective of the present invention is to provide a novel method for synthesizing 4-aminocinnamic acid.

Solution to Problem

The present inventors have made earnest studies and, as a result, have conceived that a novel route for synthesizing 4-aminocinnamic acid from glucose can be established by using 4-nitrophenylalanine, whose synthetic pathway from glucose is known, as a raw material, and converting it to 4-nitrocinnamic acid and then to 4-aminocinnamic acid. The present inventors have also conceived that both the conversion of 4-nitrophenylalanine to 4-nitrocinnamic acid and the conversion of 4-nitrocinnamic acid to 4-aminocinnamic acid can be achieved using appropriate enzymes of biological origins, thereby accomplishing the present invention.

Some aspects of the present invention relate to the following:[1] A method for producing 4-aminocinnamic acid from 4-nitrophenylalanine, comprising the steps of:(1) converting 4-nitrophenylalanine to 4-nitrocinnamic acid; and(2) converting 4-nitrocinnamic acid to 4-aminocinnamic acid.[2] The method according to [1], wherein the conversion of step (1) is carried out by using a first enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:1, 3, or 5 and which has the ability to convert 4-nitrophenylalanine to 4-nitrocinnamic acid.[3] The method according to [2], wherein the conversion of step (1) is carried out with a first host cell which has been engineered to express the first enzyme.[4] The method according to [3], wherein the first host cell is a microorganism cell.[5] The method according to [4], wherein the microorganism is a bacterium.[6] The method according to [5], wherein the conversion of step (1) is carried out via a resting-cell reaction using a resting bacterial cell as the first host cell.[7] The method according to any one of [1] to [6], wherein the conversion of step (2) is carried out by using a second enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:7, 9, 11, 13, or 15 and which has the ability to convert 4-nitrocinnamic acid to 4-aminocinnamic acid.[8] The method according to [7], wherein the conversion of step (2) is carried out by using a second host cell which expresses the second enzyme.[9] The method according to [8], wherein the second host cell is a host cell engineered to express the second enzyme.[10] The method according to [8] or [9], wherein the second host cell is a microorganism cell.[11] The method according to [10], wherein the microorganism is a bacterium.[12] The method according to [11], wherein the conversion of step (2) is carried out via a resting-cell reaction using a resting bacterial cell as the second host cell.[13] The method according to [6] or [12], wherein the resting bacterial cell is selected from the group consisting of cultured cells, powdered cells, and immobilized cells.[14] The method according to any one of [7] to [13], wherein the conversion of step (2) is carried out at a pH of from 8 to 9.[15] A method for producing 4-aminocinnamic acid from glucose, comprising the steps of:(a) producing phenylalanine from glucose;(b) converting the phenylalanine obtained in step (a) to 4-nitrophenylalanine via nitration; and(c) producing 4-aminocinnamic acid from the 4-nitrophenylalanine obtained in step (b) via a method according to any one of [1] to [14].[16] A method for producing 4-aminocinnamic acid from phenylalanine, comprising the steps of:(b) converting phenylalanine to 4-nitrophenylalanine via nitration; and(c) producing 4-aminocinnamic acid from the 4-nitrophenylalanine obtained in step (b) via a method according to any one of [1] to [14].[17] A method for producing 4-nitrocinnamic acid from 4-nitrophenylalanine, comprising:

using a first enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:1, 3, or 5 and which has the ability to convert 4-nitrophenylalanine to 4-nitrocinnamic acid.[18] A method for producing 4-aminocinnamic acid from 4-nitrocinnamic acid, comprising:

using a second enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:7, 9, 11, 13, or 15 and which has the ability to convert 4-nitrocinnamic acid to 4-aminocinnamic acid.[19] A vector carrying a nucleic acid encoding a first enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:1, 3, or 5 and which has the ability to convert 4-nitrophenylalanine to 4-nitrocinnamic acid.[20] A vector carrying a nucleic acid encoding a second enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:7, 9, 11, 13, or 15 and which has the ability to convert 4-nitrocinnamic acid to 4-aminocinnamic acid.[21] A host cell engineered to express a first enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:1, 3, or 5 and which has the ability to convert 4-nitrophenylalanine to 4-nitrocinnamic acid.[22] A host cell engineered to express a second enzyme which has an amino acid sequence having a sequence homology of 80% or more to an amino acid sequence shown in SEQ ID NOs:7, 9, 11, 13, or 15 and which has the ability to convert 4-nitrocinnamic acid to 4-aminocinnamic acid.

Advantageous Effects of Invention

The present invention provides a novel method for synthesizing 4-aminocinnamic acid from glucose.

