NOVEL BACTERIAL STRAINS FOR THE PRODUCTION OF VANILLIN

• US 20180320204A1
• Filed: 09/22/2016
• Published: 11/08/2018
• Est. Priority Date: 09/29/2015
• Status: Active Grant

• Current Assignee: Lesaffre Et Compagnie
• Original Assignee: Lesaffre Et Compagnie
• Inventors: DARRICAU, Mylene, DESFOUGERES, Thomas, PERNODET, Jean-Luc
• Granted Patent: US 10,450,591 B2
• Time in Patent Office: Days
• CPC Class Codes: C07C 47/58 Vanillin
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 7/24 containing a carbonyl group
• CPC Class Codes: C12P 7/26 Ketones
• CPC Class Codes: C12Y 401/02041 Vanillin synthase (4.1.2.41)
• CPC Class Codes: C12Y 402/01101 Trans-feruloyl-CoA hydratase (4.2.1.101)
• CPC Class Codes: C12Y 602/01034 Trans-feruloyl-CoA synthase (6.2.1.34)

Abstract

The invention relates to an Amycolatopsis strain derived from the Amycolatopsis Zyl 926 strain having at least one additional copy of the FCS and ECH genes encoding feruloyl-CoA-synthetase and enoyl-CoA-hydratase/aldolase integrated at the integration site of the φC31 phage.

The invention also relates to the method for producing such strains, the integration cassette of the FCS and ECH genes, the use of said strains in a method for producing vanillin, and a method for producing vanillin.

Claims

1. An Amycolatopsis strain derived from the Amycolatopsis Zyl 926 strain filed with the CABI Bioscience under the number IMI 390106 on 2 Mar. 2003, characterized in that it comprises at least one additional copy of the FCS gene encoding feruloyl-coA-synthetase having the protein sequence SEQ ID NO:

1 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO sequence;

1, and at least one additional copy of the ECH gene encoding Enoyl-coA Hydratase/Aldolase having the protein sequence SEQ ID NO;

2 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO sequence;

2 and in that said additional copies of the FCS and ECH genes are specifically incorporated at the φ

C31 phage integration site.

2. An Amycolatopsis strain according to claim 1, characterized in that said additional copies of the FCS and ECH genes are placed under the control of strong ermE* promoter associated with a ribosome combining site.

8. An Amycolatopsis sp. strain according to claim 1 selected from the strain filed with the CNCM under the number I-4922, the strain filed with the CNCM under the number I-4923 and the strain filed with the CNCM under the number I-4924.

12. Use in a method of producing vanillin from a Amycolatopsis sp. strain derived from the Amycolatopsis Zyl 926 strain according to claim 1.

13. A method of producing vanillin, characterized in that the bioconversion reaction is carried out in a medium comprising an Amycolatopsis sp. strain derived from the Amycolatopsis Zyl 926 strain according to claim 1.

3. A process for obtaining an Amycolatopsis strain derived from the Amycolatopsis Zyl 926 strain filed with the CABI Bioscience under the number IMI 390106 on 2 Mar. 2003, comprising at least one additional copy of the FCS gene encoding feruloyl-coA-synthetase having the sequence SEQ ID NO:

1 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO sequence;

1, and the ECH gene encoding the Enoyl-coA Hydratase/Aldolase having the sequence SEQ ID NO sequence;

2 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO sequence;

2 and of which said additional copies of FCS and ECH genes are specifically integrated at the φ

C31 phage integration site, comprising the following steps;

a. PCR amplification of the FCS gene encoding feruloyl-CoA-synthetase having the sequence SEQ ID NO;

1 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO sequence;

1 and ECH encoding enoyl-CoA-hydratase/Aldolase having sequence SEQ ID NO;

2 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO;

2, isolated from an Amycolatopsis strain;

b. constructing a vector carrying the nucleic acid sequences obtained in step a, from pSET152 plasmid.

c. transformation by electroporation of the ET12567 Escherichia coli strain with plasmid comprising combining all of the genes encoding the transfer machinery the origin of transfer oriT of which is rendered non-functional, and the vector constructed in step b;

d. selection of strains of Escherichia coli ET12567 transformed in step c by culture in a medium containing neomycin and a beta-lactam;

e. interspecific transfer between Escherichia coli ET12567 strains transformed and selected in steps c and d and the Amycolatopsis Zyl 926 strain filed with the CABI Bioscience under the number IMI 390106 on 2 Mar. 2003, for implementation in co-culture;

f. selection of Amycolatopsis Zyl 926 strains having integrated at least one additional copy of the FCS gene encoding feruloyl-coA-synthetase having the sequence SEQ ID NO;

1 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO sequence;

1 and at least one additional copy of the ECH gene encoding enoyl-coA-hydratase/aldolase having the sequence SEQ ID NO sequence;

2 or sequence at least 80/%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO;

2 according to step e, by culturing in a medium containing apramycin.

4. A method according to claim 3, characterized in that it comprises an additional step b′

between steps b and c, of amplification and cloning of the strong ermE* promoter having the nucleic acid sequence of SEQ ID NO;

6 or any sequence at least 90%, preferably at least 95% identical to the sequence SEQ ID NO;

6 into the vector constructed in step b, said ermE* promoter is associated with a ribosome binding site.

5. A method according to claim 4, characterized in that the conjugating plasmid of step c is the pUZ8002 plasmid.

6. Method according to claim 3, characterized in that the beta-lactam from step d is ampicillin.

7. An Amycolatopsis sp. strain derived from the Amycolatopsis Zyl 926 strain obtainable by the process according to claim 3.

9. An integration cassette characterized in that it comprises:

i. an expression cassette of the FCS gene encoding feruloyl-coA-synthetase having the sequence SEQ ID NO;

1 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO;

1 and ECH encoding enoyl-coA-hydratase/aldolase having the sequence SEQ ID NO;

2 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to SEQ ID NO;

2;

ii. means for integration of the cassette into the genome of the strain Amycolatopsis Zyl926, comprising the int gene of the φ

C31 phage having the sequence SEQ ID NO;

3 or any sequence at least 80%, preferably at least 90%, and more preferably at least 95% identical to the sequence SEQ ID NO;

3 and the attP attachment site of the φ

C31 phage having the sequence SEQ ID NO;

4;

iii. means for selecting transformed Escherichia coli ET12567 strains comprising neomycin and a beta-lactam;

iv. means for selecting Amycolatopsis Zyl926 strains having integrated said integration cassette comprising a marker for resistance to apramycin.

10. An integration cassette according to claim 9, characterized in that the FCS and ECH genes encoding feruloyl-coA-synthetase and enoyl-coA-hydratase/aldolase of the expression cassette are under the control of the ermE* promoter having the sequence SEQ ID NO:

6 or any sequence at least 90%, and preferably at least 95% identical to SEQ ID NO;

6, said ermE* promoter associated with a ribosome binding site.

11. An integration cassette according to claim 9, characterized in that the beta-lactam antibiotic is ampicillin.