Diphosphomevalonate decarboxylase variant and method for manufacturing olefin compound using same

Patent metadata

Field	Value
Publication number	US-10988751-B2
Application number	US-201816481585-A
Country	US
Kind code	B2
Filing date	Jan 30, 2018
Priority date	Feb 1, 2017
Publication date	Apr 27, 2021
Grant date	Apr 27, 2021

Abstract

A method enables olefin compound production with a high productivity, and an enzyme is used in the method. Mutations involving amino acid substitution are introduced into various sites of diphosphomevalonate decarboxylase (MVD) to prepare multiple MVD variants. Then, these variants are evaluated in terms of catalytic activity for producing an olefin compound such as isoprene, and have found as a result that the catalytic activity is improved when serine at position 153 and threonine at position 209 are each substituted with a different amino acid. In addition, the catalytic activity of the variants is further improved when glycine at position 152 is further substituted with a different amino acid.

First claim

The invention claimed is:

1. A method for producing isoprene or isobutene, the method comprising:
reacting ATP and a substrate compound in the presence of a diphosphomevalonate decarboxylase to produce isoprene or isobutene,
wherein:
the diphosphomevalonate decarboxylase is a protein in which:
serine at position 153 of SEQ ID NO: 2 or serine corresponding to the position is mutated to aspartic acid or glutamic acid,
threonine at position 209 of SEQ ID NO: 2 or threonine corresponding to the position is mutated to aspartic acid, and
the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which no more than 40 amino acid residues of the amino acid sequence of SEQ ID NO: 2 are substituted, deleted, added, and/or inserted other than at positions 153 and 209,

and
when the method produces isoprene, the substrate compound is 3-hydroxy-3-methylpent-4-enoate and
when the method produces isobutene, the substrate compound is (β-hydroxyisovaleric acid.

2. The production method according to claim 1 , wherein reacting ATP and the substrate compound in the presence of the diphosphomevalonate decarboxylase is performed by culturing a host cell comprising:
a DNA encoding the diphosphomevalonate decarboxylase, or
a vector comprising the DNA.

3. The production method according to claim 1 , wherein in the diphosphomevalonate decarboxylase, glycine at position 152 of SEQ ID NO: 2 or glycine corresponding to the position is mutated to a different amino acid.

4. The production method according to claim 3 , wherein the different amino acid mutated from the glycine at position 152 of SEQ ID NO: 2 or the glycine corresponding to the position is leucine.

5. The production method according to claim 1 , wherein the method produces isoprene.

Assignees

• Riken
• Zeon Corp
• Yokohama Rubber Co Ltd

Inventors

• ORISHIMO Ryoko
• Shirai Tomokazu
• Takahashi Kazuhiro
• Hiza Misao
• Tanabe Yusuke

Classifications

• C12P5/026 Primary

  Unsaturated compounds, i.e. alkenes, alkynes or allenes · CPC title

• C12P5/02

  acyclic {(C12P5/007 takes precedence)} · CPC title

• C12Y401/01033

  Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12P5/007

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title