Modified isoprene synthase

Patent metadata

Field	Value
Publication number	US-2016257944-A1
Application number	US-201615159140-A
Country	US
Kind code	A1
Filing date	May 19, 2016
Priority date	Nov 22, 2013
Publication date	Sep 8, 2016
Grant date	—

Abstract

Modified isoprene synthases that have one or more mutations of a given amino acid residue(s) in any amino acid sequence of
(a) the amino acid sequence of SEQ ID NO:4, (b) an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO:4, or (c) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:4,

and has an isoprene synthetic activity are useful for preparing isoprene monomer in improved yields.

First claim

1 . A modified isoprene synthase that has one or more mutations of an amino acid residue corresponding to one or more amino acid residues selected from the following:
S2, F31, L35, L67, I90, V97, L114, Q123, D124, V125, K127, R128, K130, D131, G134, C137, V144, V187, R202, L224, L247, S248, L249, T257, K259, R264, D265, R266, E269, C286, K292, T298, I300, D301, Y304, D305, E312, F316, E321, V325, I328, C338, L340, C370, E371, C373, E379, S383, K386, I388, A390, Y394, S401, S402, G404, V405, L414, C416, C440, R444, N447, R458, T461, T462, T466, E471, C480, K481, R484, K492, V499, P505, F508, I518, S519, H520, C521, Y523, and G530 in any amino acid sequence of (a) the amino acid sequence of SEQ ID NO:4, (b) an amino acid sequence having one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO:4, or (c) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:4, and has an isoprene synthetic activity.

2 . The modified isoprene synthase according to claim 1 , wherein said mutation is selected from the group consisting of S2A, F31E, F31K, L35E, L35K, L67E, 190E, V97E, L114E, Q123C, D124E, V125E, K127D, K127E, K127N, K127R, R128K, K130G, D131C, G134P, C137E, C137I, C137L, C137M, C137S, V144E, V187E, R202D, R202N, L224E, L247D, L247E, L247Q, S248E, S248K, S248R, L249E, L249K, L249R, L249T, T257L, K259D, K259E, K259N, K259Q, K259R, R264F, R264M, R264T, D265E, D265N, D265Q, D265R, R266N, R266Q, E269D, E269I, E269Y, C286V, C286N, K292L, T298I, I300N, D301R, Y304F, D305R, E312I, E312R, F316E, E321D, V325K, I328E, C338V, C338L, L340K, C370E, C370N, C370K, C370V, E371D, C373V, C373Y, E379T, S383I, K386R, I388C, A390C, Y394F, S401T, S402L, G404A, G404M, V405C, L414C, C416E, C440S, R444T, N447L, N447R, R458Q, T461R, T462M, T462S, T466F, T466C, T466W, T466M, T466Y, T466H, T466P, T466Q, T466N, T466A, E471C, C480N, C480Y, C480V, K481E, R484K, K492E, V499I, P505H, F508C, F508Q, F508R, I518C, I518S, S519N, H520N, C521N, C521S, C521V, C521E, C521I, Y523I, Y523L, and G530K.

3 . The modified isoprene synthase according to claim 1 , further having a signal sequence at N-terminus.

4 . The modified isoprene synthase according to claim 3 , which has one or more mutations of an amino acid residue corresponding to one or more amino acid residues selected from the following:
S45, F74, L78, L110, I133, V140, L157, Q166, D167, V168, K170, R171, K173, D174, G177, C180, V187, V230, R245, L267, L290, S291, L292, T300, K302, R307, D308, R309, E312, C329, K335, T341, I343, D344, Y347, D348, E355, F359, E364, V368, I371, C381, L383, C413, E414, C416, E422, S426, K429, I431, A433, Y437, S444, S445, G447, V448, L457, C459, C483, R487, N490, R501, T504, T505, T509, E514, C523, K524, R527, K535, V542, P548, F551, I561, S562, H563, C564, Y566, and G573 in any amino acid sequence of (d) the amino acid sequence of SEQ ID NO:2, (e) an amino acid sequence having one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NO:2, or (f) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:2, and has an isoprene synthetic activity.

5 . The modified isoprene synthase according to claim 4 , wherein said mutation is selected from the group consisting of S45A, F74E, F74K, L78E, L78K, L110E, I133E, V140E, L157E, Q166C, D167E, V168E, K170D, K170E, K170N, K170R, R171K, K173G, D174C, G177P, C180E, C180I, C180L, C180M, C180S, C180M, V187E, V230E, R245D, R245N, L267E, L290D, L290E, L290Q, S291E, S291K, S291R, L292E, L292K, L292R, L292T, T300L, K302D, K302E, K302N, K302Q, K302R, R307F, R307M, R307T, D308E, D308N, D308Q, D308R, R309N, R309Q, E312D, E312I, E312Y, C329V, C329N, K335L, T341I, I343N, D344R, Y347F, D348R, E355I, E355R, F359E, E364D, V368K, I371E, C381V, C381L, L383K, C413E, C413N, C413K, C413V, E414D, C416V, C416Y, E422T, S426I, K429R, I431C, A433C, Y437F, S444T, S445L, G447A, G447M, V448C, L457C, C459E, C483S, R487T, N490L, N490R, R501Q, T504R, T505M, T505S, T509F, T509C, T509W, T509M, T509Y, T509H, T509P, T509Q, T509N, T509A, E514C, C523N, C523Y, C523V, K524E, R527K, K535E, V542I, P548H, F551C, F551Q, F551R, I561C, I561S, S562N, H563N, C564N, C564S, C564V, C564E, C564I, Y566I, Y566L, and G573K.

6 . A polynucleotide encoding a modified isoprene synthase according to claim 1 .

7 . An expression vector comprising a polynucleotide according to claim 6 .

8 . A host cell comprising an expression unit of a polynucleotide encoding a modified isoprene synthase according to claim 1 .

9 . The host cell according to claim 8 , wherein said host cell has an ability to synthesize dimethylallyl diphosphate via a methylerythritol phosphate pathway.

10 . The host cell according to claim 9 , wherein said host cell is an Escherichia coli.

11 . The host cell according to claim 8 , wherein said host cell has an ability to synthesize dimethylallyl diphosphate via both a mevalonate pathway and a methylerythritol phosphate pathway.

12 . The host cell according to claim 8 , wherein said host cell is a microorganism belonging to the genus Corynebacterium , the genus Pantoea , the genus Enterobacter or the genus Saccharomyces.

13 . A method for producing a modified isoprene synthase, comprising culturing a host cell according to claim 8 .

14 . A method for producing isoprene monomer, comprising producing said isoprene monomer from dimethylallyl diphosphate in the presence of a modified isoprene synthase according to claim 1 .

15 . The method according to claim 14 , wherein said isoprene monomer is produced by cultivation of a host cell according to claim 8 in a medium.

16 . The method according to claim 15 , wherein said dimethylallyl diphosphate is supplied from a carbon source in said medium by cultivation of said host cell.

17 . A method for producing an isoprene polymer, comprising:
(I) producing an isoprene monomer by the method according to claim 14 ; and (II) polymerizing said isoprene monomer to form said isoprene polymer.

18 . A method for producing a rubber composition, comprising:
(A) preparing an isoprene polymer by the method of claim 17 ; and (B) mixing said isoprene polymer with one or more rubber composition components.

19 . A method for producing a tire, comprising:
(i) producing a rubber composition by the method of claim 18 ; and (ii) forming said rubber composition into a tire.

Assignees

• Ajinomoto Kk

Inventors

• Nakata Kunio
• Tajima Yoshinori
• Tagami Uno
• Oku Takashi
• Kashima Yasuhiro

Classifications

• C12Y402/03027

  Isoprene synthase (4.2.3.27) · CPC title

• C12P5/007

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

• C08F36/08

  Isoprene · CPC title

• C12Y402/03007

  Pentalenene synthase (4.2.3.7) · CPC title

• C12P5/026

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