Use of the ssDNA Recombineering technique to knock out the manitol dehydrogenase gene in Lactobacillus reuteri CRL 1101

JULIANA BLECKWEDEL; MARIA EUGENIA ORTIZ; FERNANDA MOZZI; RAÚL R. RAYA

San Miguel de Tucumán

Simposio; IV Simposio Internacional de Bacterias Lácticas; 2013

Centro de referencia para lactobacilos

Mannitol is a polyol widely used in the pharmaceutical, chemical, and food industries while in medicine it is employed as a potent osmotic diuretic. Mannitol production by lactic acid bacteria (LAB) has several advantages as compared with chemical synthesis such as a complete conversion of D-fructose into D-mannitol without co-formation of sorbitol; mild production conditions; and no requirement of highly purified substrates. Lactobacillus reuteri CRL1101 produces mannitol from sugarcane molasses, a low-cost substrate rich in sucrose and fructose, being the enzyme mannitol-2-dehydrogenase (E.C. 1.1.1.138) (MDH) responsible for the conversion of fructose into mannitol. The mdh gene encompasses 1,011-bp and encodes a protein consisting of 336 amino acids (aa) with a predicted MM of 35,920 Da. In this work, a double-stop codon mutation was introduced into the mdh gene of strain CRL 1101 by using the ssDNA recombineering technique described by van Pijkeren and Britton (2012). In our hands, rifampicin-resistat cell mutants (tested with an oligonucleotide targeting the rpoB gene) were recovered at a frequency of 2.5%. CRL 1101 cells were electroporated with an 81-mer oligonucleotide carrying two stop codons 174-nt downstream of the mdh initiation codon. Potential 58 aa nonfunctional MDH cells were plated without selection in MRS agar. Positive mutants were identified by the MAMA-PCR technique. Similar specific MDH activities for both the wt and mutant strains in both media (MRS Glu7%: 0,87-0,67 and MRS Glu2%+Fru5%: 2,37-2,83) were observed. These results suggest the presence of more than one gene with MDH activity into the CRL 1101 chromosome.