Recombinant strain producing o-aminobenzoate and fermentative production of aniline from renewable resources via 2-aminobenzoic acid

Patent metadata

Field	Value
Publication number	US-2017152535-A1
Application number	US-201515119825-A
Country	US
Kind code	A1
Filing date	Feb 19, 2015
Priority date	Feb 20, 2014
Publication date	Jun 1, 2017
Grant date	—

Abstract

The invention provides a recombinant microbial host cell capable of converting a raw material comprising a fermentable carbon substrate to o-aminobenzoate biologically. The invention further provides a method for producing aniline, comprising the steps of: a) producing o-aminobenzoate by fermentation of a raw material comprising at least one fermentable carbon substrate using the recombinant microbial host cell of the capable of converting said raw material comprising at least one fermentable carbon substrate to o-aminobenzoate biologically, wherein said o-aminobenzoate comprises anthranilate anion, b) converting said o-aminobenzoate from said anthranilate anion to anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolving in an organic solvent, and d) converting said anthranilic acid to aniline by thermal decarboxylation in an organic solvent.

First claim

1 . A recombinant microbial host, cell capable of converting a raw material comprising a fermentable carbon substrate to o-arninobenzoate biologically.

2 . The recombinant microbial host cell of claim 1 , wherein said microbial host cell is selected from the group consisting of bacteria, yeast and fungi.

3 . The recombinant microbial host cell of claim 26 , wherein said Corynebacterium glutamicum comprises a genetic modification of the trpD gene (SEQ ID NO: 1) encoding anthranilate phosphoribosyl transferase, and wherein said genetic modification has the effect of a reduced expression of the trpD gene,

4 . The recombinant microbial host cell of claim 26 , wherein said Corynebacterium glutamicum comprises a genetic modification of the csm gene encoding chorismate mutase (SEQ ID NO: 9), and wherein said genetic modification has the effect of a reduced expression of the csm gene.

5 . The recombinant microbial host cell of claim 26 , wherein said Corynebacterium glutamicum microbial host cell further comprises one or more deletions selected from the group consisting of fpr (SEQ ID NO:17 or SEQ ID NO: 18), ptsG (SEQ D NO:19 or SEQ iD NO:20), pepco (SEQ ID NO:21 or SEQ ID NO:22), pyk (SEQ ID NO: 23 or SEQ ID NO: 24), and gpi (SEQ. ID NO: 27 or SEQ ID NO: 28).

6 . The recombinant microbial host cell of claim 26 , wherein said Corynebacterium glutamicum microbial host cell further overexpresses one or more of the genes selected from the group consisting of galP (SEQ ID NO: 30), loIT2 (SEQ ID NO: 31), ppgk (SEQ ID NO: 32), pps (SEQ ID NO: 33-35), ppk. (SEQ ID NO: 36), zwf1 (SEQ ID NO: 37), opcA (SEQ ID NO: 38-39), tktCG (SEQ 1D NO: 40), tktEC (SEQ ID NO: 41), talCG (SEQ ID NO: 42), talEC (SEQ ID NO: 43), qsuA (SEQ ID NO: 96), trpEGS38F (SEQ ID NO: 50), trpEGS33R. (SEQ ID NO: 51), trpEGS40R (SEQ D NO: 52), trpEGS40F (SEQ ID NO: 53), aroGD146N (SEQ ID NO: 55), aroL, (SEQ ID NO: 93), aroK (SEQ ID NO: 94), and glnA (SEQ ID NO: 95).

7 . The recombinant microbial host cell of claim 28 , wherein said Pseudomonas putida comprises a deletion of the trpDC gene encoding anthranilate phosphoribosyl transferase (SEQ ID NO: 63 and SEC) ID NO: 65), or a deletion of the pheA gene encoding chorismate mutase (SEQ ID NO: 64 and SEQ ID NO 65), or both.

8 . The recombinant microbial host cell of claim 28 , wherein said Pseudomonas putida overexpresses one or more of the genes selected from the group consisting trpEGS40F ‘SEQ ID NO: 53) and aroGD146N (SEQ ID NO: 55).

9 . The recombinant microbial host cell of claim 6 , wherein said one or more overexpressed genes are integrated into said Corynebacterium giutamicum microbial host cell by plasmid transformation or by chromosomal transformation,

10 . The recombinant microbial host cell of claim 1 , wherein said raw material is selected from the group consisting of sugar beet, sugar cane, starch-containing plants, lignocellulose, glycerol and C1-compound.

11 . The recombinant microbial host cell of claim 1 , wherein said fermentable carbon substrate is selected from the group consisting of C-5 monosaccharides, C-6 monosaccharides, disaccharides, and tri-saccharides.

12 . A method for producing aniline, comprising:
a) producing o-aminobenzoate by fermentation of a raw material comprising at least one fermentable carbon substrate using the recombinant microbial host cell of claim 1 , wherein said o-aminobenzoate comprises anthranilate anion, b) converting said o-aminobenzoate from said an.hranilate anion to anthranilic acid by acid protonation, c) recovering said anthranilic acid by precipitation or by dissolving in an organic solvent, and d) converting said anthranilic acid to anil ne by thermal decarboxylation in an organic solvent.

13 . The method of claim 12 , wherein said fermentation of step a) is a batch fermentation, a fed-batch fermentation or a continuous fermentation.

14 . The method of claim 12 , wherein at least step a) and step b) are run continuously,

15 . The method of claim 12 , wherein said recombinant microbial host is removed prior to performing step b) of converting said o-aminobenzoate from said anthranilate anion to anthranilic acid.

16 . The method of claim 12 , wherein said acid protonation of step b) is done by adding HCl.

17 . The method of claim 12 , wherein in step c) said recovering of said anthranilic acid by precipitation comprises filtration, thereby generating a slurry comprising said recovered anthranilic acid.

18 . The method of claim 12 , wherein in step c) said recovering by dissolving said anthranilic acid in an organic solvent comprises adding said organic solvent, to said anthranilic acid such that said anthranilic acid is recovered as a solute in said organic solvent.

19 . The method of claim 12 , wherein step c) is followed by washing and drying the recovered anthranilic acid precipitate advance of performing the thermal decarboxylation of step d).

20 . The method of claim 12 , wherein step d) is performed in the presence of a catalyst.

21 . The method of claim 12 , wherein follovving step c) residual anthranilate anion is recovered by adsorption to an ion exchange resin or an active carbon material or a zeolite, followed by desorption of the recovered anthranilate anion.

22 . The method according to claim 21 , wherein subsequent to the recovery of the residual anthranilate anion by adsorption following step c), a water stream devoid of the adsorbed anthranilate anion is at least partially re-fed to the fermentation of step a).

23 . A method of using aniline produced by the method of claim 12 comprising converting the aniline to methylenedianiline (MDA) with formaldehyde in the presence of water and catalyst.

24 . A method of using the MDA produced by the method of claim 23 , comprising converting the MDA to methyienediisocyanate (MDI) with phosgene.

25 . The recombinant microbial host cell of claim 2 . wherein said microbial host cell is bacteria, wherein said bacterium is an Escherichia coli strain, a Corynebacterium strain or a Pseudomonas strain.

26 . The recombinant microbial host cell of claim 25 , wherein said bacterium is a Corynebacterium strain, wherein said Corynebacterium strain is Corynebacterium glutamicum.

27 . The recombinant microbial host cell of claim 26 , wherein said Corynebacterium glutarnicum is Corynebacterium glutamicum ATCC 13032.

28 . The recombinant microbial host cell of claim 25 , wherein said bacterium is a Pseudomonas strain, wherein said Pseudomonas strain is Pseudomonas putida.

29 . The recombinant microbial host cell of claim 28 wherein said Pseudomonas putida is Pseudomonas putida KT2440.

30 . The recombinant microbial host cell of claim 3 , wherein said Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.

31 . The recombinant microbial host cell of claim 3 , wherein said genetic modification is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ. ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:8.

32 . The recombinant microbial host cell of claim 4 , wherein said Corynebacterium glutamicum microbial host cell further comprises one or more deletions selected from the group consisting of hpr (SEQ ID NO:17 or SEQ ID NO: 18), ptsG (SEQ ID NO:19 or SEQ ID

Assignees

• Covestro Deutschland Ag

Inventors

• Jaeger Gernot
• Magnus Jorgen
• Moussa Amgad Salah
• Olf Guenter
• Lolli Giulio
• Behnken Swantje
• Youn Jung-Won
• Yalfani Mohammad
• Sprenger Georg

Classifications

• C12P13/001 Primary

  Amines; Imines · CPC title

• C12N9/1077 Primary

  Pentosyltransferases (2.4.2) · CPC title

• C12N15/77 Primary

  for Corynebacterium; for Brevibacterium · CPC title

• C12Y204/02018

  Anthranilate phosphoribosyltransferase (2.4.2.18) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title