Use of the ssDNA recombineering technique to knock out the mannitol dehydrogenase gene in Lactobacillus reuteri CRL 1101

J. BLECKWEDEL; M.E. ORTIZ; F. MOZZI; R.R. RAYA

San Miguel de Tucumán

Simposio; IV Simposio Internacional de Bacterias Lácticas (SIBAL). Alimentos, Salud y Aplicaciones; 2013

CERELA-CONICET

Mannitol is a polyol produced by certain plants, algae, mushrooms, yeasts, and bacteria. It is widely used in the pharmaceutical, chemical, and food industries while in medicine it is used 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. We have previously shown that Lactobacillus reuteri CRL 1101 successfully produced 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 gene encoding MDH encompasses 1,011-bp and encodes a protein consisting of 336 amino acids (aa), with a predicted molecular mass 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 (to produce a 58 aa nonfunctional MDH), and plated without selection in MRS agar. Mutants that incorporated into the chromosome the mutated primer were identified by the MAMA-PCR technique. However, although two of these mutants showed lower specific MDH activities (0,316 U/mg prot) than that (0,722 U/mg prot) of the wild type when grown all the strains in MRS containing 7% (w/v) glucose, similar specific MDH activities (1,3 ? 1,4 U/mg prot) were observed for both the wild type and mutant cells in MRS broth with glucose 2% plus fructose 5%. These results suggest the presence of more than one gene with MDH activity into the CRL 1101 chromosome. Current research is being conducted to confirm this hypothesis.