Bacterial strains for the production of vanillin

A method for obtaining an Amycolatopsis strain obtained after transformation of the Amycolatopsis Zyl 926 strain filed with the CABI Bioscience under the number IMI 390106 on 2 Mar. 2003 by interspecific transfer by integrating, at least one additional copy of the FCS gene and at least one additional copy of the ECH gene, the FCS gene encoding feruloyl-CoA-synthetase having the sequence SEQ ID NO:

• 1 or any sequence encoding feruloyl-CoA-synthetase at least 95% identical to SEQ ID NO:
• 1, and the ECH gene encoding enoyl-coA hydratase/aldolase having the sequence ID NO:
• 2 or any sequence encoding enoyl-CoA hydratase/aldolase at least 95% identical to SEQ ID NO:
• 2 and wherein said additional copies of FCS and ECH genes are specifically integrated at the φC31 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 encoding feruloyl-CoA-synthetase at least 95% identical to SEQ ID NO:
• 1 and of the ECH gene encoding enoyl-CoA hydratase/aldolase having sequence SEQ ID NO:
• 2 or any sequence encoding enoyl-CoA hydratase/aldolase 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 integratedat least one additional copy of the FCS gene encoding feruloyl-CoA-synthetase having the sequence SEQ ID NO:
• 1 or any sequence encoding feruloyl-CoA-synthetase having at least 95% identity to SEQ ID NO:
• 1 and at least one additional copy of the ECH gene encoding enoyl-CoA hydratase/aldolase having the sequence SEQ ID NO:
• 2 or any sequence encoding enoyl-CoA hydratase/aldolase having at least 95% identity to SEQ ID NO:
• 2 according to step e, by culturing in a medium containing apramycin.

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 encoding feruloyl-CoA-synthetase at least 95% identical to SEQ ID NO:
• 1, and of the ECH gene encoding enoyl-CoA hydratase/aldolase having the sequence SEQ ID NO:
• 2 or any sequence encoding enoyl-CoA hydratase/aldolase 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 φC31 phage having the sequence SEQ ID NO:
• 3 and the attP attachment site of the φC31 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 Zyl 926 strains having integrated said integration cassette comprising a marker for resistance to apramycin.