BIOTECHNOLOGICAL PRODUCTION OF omega-FUNCTIONALISED CARBOXYLIC ACIDS AND ESTERS THEREOF

Patent metadata

Field	Value
Publication number	US-2017145448-A1
Application number	US-201615359932-A
Country	US
Kind code	A1
Filing date	Nov 23, 2016
Priority date	Nov 25, 2015
Publication date	May 25, 2017
Grant date	—

Abstract

There is provided a microbial cell for producing at least one omega-functionalized carboxylic acid ester from at least one alkane, wherein the cell is genetically modified to increase the expression relative to the wild type cell of
(i) Enzyme E 1 capable of converting the alkane to the corresponding 1-alkanol; (ii) Enzyme E 2 capable of converting the 1-alkanol of (i) to the corresponding 1-alkanal; (iii) Enzyme E 3 capable of converting the 1-alkanal of (ii) to the corresponding alkanoic acid; (iv) Enzyme E 4 capable of converting the alkanoic acid of (iii) to the corresponding alkanoic acid ester; and (iv) Enzyme E 5 capable of converting the alkanoic acid ester of (iv) to the corresponding omega-hydroxy-alkanoic acid ester, and wherein the cell does not comprise a genetic modification that increases the expression relative to the wild type cell of at least one of the following enzymes E 20 -E 24 selected from the group consisting of:
E 20 Acyl-ACP thioesterase, of EC 3.1.2.14 or EC 3.1.2.22, E 21 Acyl-CoA thioesterase, of EC 3.1.2.2, EC 3.1.2.18, EC 3.1.2.19, EC 3.1.2.20 or EC 3.1.2.22, E 22 Acyl-CoA:ACP transacylase, E 23 Polyketide synthase, and E 24 Hexanoic acid synthase

First claim

1 . A microbial cell for producing at least one ω-functionalized carboxylic acid ester from at least one alkane, wherein the cell is genetically modified to increase the expression relative to the wild type cell of
(i) Enzyme E 1 capable of converting the alkane to the corresponding 1-alkanol;
(ii) Enzyme E 2 capable of converting the 1-alkanol of (i) to the corresponding 1-alkanal;
(iii) Enzyme E 3 capable of converting the 1-alkanal of (ii) to the corresponding alkanoic acid;
(iv) Enzyme E 4 capable of converting the alkanoic acid of (iii) to the corresponding alkanoic acid ester; and
(v) Enzyme E 5 capable of converting the alkanoic acid ester of (iv) to the corresponding ω-hydroxy-alkanoic acid ester,
and wherein the cell does not comprise a genetic modification that increases the expression relative to the wild type cell of at least one of the following enzymes E 20 -E 24 selected from the group consisting of:
E 20 Acyl-ACP thioesterase, of EC 3.1.2.14 or EC 3.1.2.22,
E 21 Acyl-CoA thioesterase, of EC 3.1.2.2, EC 3.1.2.18, EC 3.1.2.19, EC 3.1.2.20 or EC 3.1.2.22,
E 22 Acyl-CoA:ACP transacylase,
E 23 Polyketide synthase, and
E 24 Hexanoic acid synthase.

2 . The cell according to claim 1 , wherein the cell is further genetically modified to increase the expression relative to the wild type cell of
(vi) Enzyme E 6 capable of converting the corresponding ω-hydroxy-alkanoic acid ester of (v) to the corresponding ω-oxo alkanoic acid ester; or (vii) Enzyme E 6 capable of converting the corresponding ω-hydroxy-alkanoic acid ester of (v) to the corresponding ω-oxo alkanoic acid ester and Enzyme E 7 capable of converting the ω-oxo alkanoic acid ester to the corresponding ω-amino alkanoic acid ester; or (viii) Enzyme E 6 capable of converting the corresponding ω-hydroxy-alkanoic acid ester of (v) to the corresponding ω-oxo alkanoic acid ester and Enzyme E 13 capable of converting the ω-oxo alkanoic acid ester to the corresponding ω-carboxy alkanoic acid ester and Enzyme E 14 capable of converting the ω-carboxy alkanoic acid ester to the corresponding ω-carboxy alkanoic acid diester.

3 . The cell according to either claim 1 , wherein
the Enzyme E 1 is selected from the group consisting of P450 alkane hydroxylases (E a ) of EC 1.14.15.3- and AlkB alkane hydroxylases (E b ) of EC 1.14.15.3; the Enzyme E 2 is selected from the group consisting of P450 alkane hydroxylases (E a ) of EC 1.14.15.3-, AlkB alkane hydroxylases (E b ) of EC 1.14.15.3, alcohol oxidases (E c ) of EC 1.1.3.20 and alcohol dehydrogenases (E d ) of EC 1.1.1.1 or EC 1.1.1.2; the Enzyme E 3 is selected from the group consisting of P450 alkane hydroxylases (E a ) of EC 1.14.15.3-, AlkB alkane hydroxylases (E b ) of EC 1.14.15.3, aldehyde dehydrogenases (E e ), bifunctional alcohol oxidases (E c ) of EC 1.1.3.20, bifunctional AlkJ alcohol dehydrogenases (E di ) and bifunctional alcohol dehydrogenases (E dii ) of EC 1.1.1.1 or EC 1.1.1.2, wherein E c , E di , and E dii are capable of oxidizing an ω-hydroxy alkanoic acid ester directly to the corresponding ω-carboxy alkanoic acid ester; the Enzyme E 4 is selected from the group consisting of wax-ester synthases (E f ) of EC 2.3.1.75 or alcohol O-acyl transferases (E g ) of EC 2.3.1.84; the Enzyme E 5 is selected from the group consisting of P450 alkane hydroxylases (E a ) of EC 1.14.15.3- and AlkB alkane hydroxylases (E b ) of EC 1.14.15.3; the Enzyme E 6 is selected from the group consisting of P450 alkane hydroxylases (E a ) of EC 1.14.15.3-, AlkB alkane hydroxylases (E b ) of EC 1.14.15.3, alcohol oxidases (E c ) of EC 1.1.3.20, aldehyde dehydrogenases (E e ) and alcohol dehydrogenases (E d ) of EC 1.1.1.1 or EC 1.1.1.2; when present the Enzyme E 7 is an ω-transaminase (E h ) EC 2.6.1; when present the Enzyme E 13 is selected from the group consisting of P450 alkane hydroxylases (E a ) of EC 1.14.15.3-, AlkB alkane hydroxylases (E b ) of EC 1.14.15.3, aldehyde dehydrogenases (E e ), bifunctional alcohol oxidases (E c ) of EC 1.1.3.20, bifunctional AlkJ alcohol dehydrogenases (E di ) and bifunctional alcohol dehydrogenases (E diii ) of EC 1.1.1.1 or EC 1.1.1.2, wherein E c , E di , and E dii are capable of oxidizing an ω-hydroxy alkanoic acid ester via an ω-oxo alkanoic acid ester directly to the corresponding ω-carboxy alkanoic acid ester; and when present the Enzyme E 14 is selected from the group consisting of wax-ester synthases (E f ) of EC 2.3.1.75 or alcohol O-acyl transferases (E g ) of EC 2.3.1.84.

4 . The cell according to claim 1 , wherein the Enzymes E 1 , E 2 , E 3 , E 5 and E 6 , is at least one AlkB alkane hydroxylase (E b ) and Enzyme E 4 is a wax-ester synthase (E f ).

5 . The cell according to claim 4 , wherein
the AlkB alkane hydroxylase (E b ) comprises at least 60% sequence identity relative to SEQ ID NO:1; the wax-ester synthase (E f ) comprises at least 60% sequence identity relative to SEQ ID NO:2; and when present the ω-transaminase (E h ) comprises at least 60% sequence identity relative to SEQ ID NO:3.

6 . The cell according to claim 1 , wherein the cell is further genetically modified to increase the expression relative to the wild type cell of at least one acyl-CoA synthetase (E 12 ) of EC 6.2.1.3 or EC 2.3.1.86.

7 . A method of producing at least one ω-functionalized carboxylic acid ester, wherein the method comprises a step of contacting at least one cell according to claim 1 with at least one alkane.

8 . The method according to claim 7 , wherein
the ω-functionalized carboxylic acid ester is 12-amino lauric acid methyl ester, 12-hydroxy lauric acid methyl ester, 12-carboxy lauric acid methyl (di) ester and/or lauric acid methyl ester from the alkane dodecane; and/or the ω-functionalized carboxylic acid ester is 11-amino undecanoic acid methyl ester, 11-hydroxy undecanoic acid methyl ester, 11-carboxy undecanoic acid methyl (di) ester and/or undecanoic acid methyl ester from the alkane undecane.

9 . A method of producing at least one ω-functionalized carboxylic acid ester from an alkane, wherein the method comprises:
(a) contacting the following enzymes with the alkane:
(i) Enzyme E 1 capable of converting the alkane to the corresponding 1-alkanol;
(ii) Enzyme E 2 capable of converting the 1-alkanol of (i) to the corresponding 1-alkanal;
(iii) Enzyme E 3 capable of converting the 1-alkanal of (ii) to the corresponding alkanoic acid;
(iv) Enzyme E 4 capable of converting the alkanoic acid of (iii) to the corresponding alkanoic acid ester; and
(vi) Enzyme E 5 capable of converting the alkanoic acid ester of (iv) to the corresponding ω-hydroxy-alkanoic acid ester.

10 . The method according to claim 9 , wherein the method comprises
(b) contacting the following enzymes with the ω-hydroxy-alkanoic acid ester:
(vi) Enzyme E 6 capable of converting the corresponding ω-hydroxy-alkanoic acid ester of (v) to the corresponding ω-oxo alkanoic acid ester; or
(vii) Enzyme E 6 capable of converting the corresponding ω-hydroxy-alkanoic acid ester of (v) to the corresponding ω-oxo alkanoic acid ester and Enzyme E 7 capable of converting the ω-oxo alkanoic acid ester to the corresponding ω-amino alkanoic acid ester; or
(viii) Enzyme E 6 capable of converting the corresponding ω-hydroxy-alkanoic acid ester of (v) to the corresponding ω-oxo alkanoic acid ester and Enzyme E 13 capable of converting the ω-oxo alkanoic acid ester to the corresponding ω-carboxy alkanoic acid ester and Enzyme E 14 capable of converting the ω-carboxy alkanoic acid ester of (vi) to the corresponding ω-carboxy alkanoic acid diester.

11 . The method according to claim 9 , wherein
the Enzyme E 1 is selected

Assignees

• Evonik Degussa Gmbh

Inventors

• Schaffer Steffen
• Haas Thomas
• Bruegging Wilhelm
• Meier Ralf

Classifications

• C12Y206/01

  Transaminases (2.6.1) · CPC title

• C12N9/0077

  with a reduced iron-sulfur protein as one donor (1.14.15) · CPC title

• C12Y101/01002

  Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12Y101/01001

  Alcohol dehydrogenase (1.1.1.1) · CPC title