Characterization of mannitol activity and mannitol production in mannitol dehydrogenase and lactate dehydrogenase mytants of Fructobacillus tropaeoli CRL 2034

J. BLECKWEDEL; R. R. RAYA; F. MOZZI

Simposio; V Simposio Internacional de Bacterias Lácticas; 2016

CERELA

Mannitol is a sugar alcohol claimed to have health-promoting properties and widely used in the food, chemical and pharmaceutical industries. In the food industry, mannitol is employed as a non-metabolizable, low-calorie, insulin independent sweetener applicable in light and diabetic food products. Certain plants, algae, mushrooms, yeasts, and lactic acid bacteria (LAB) are known to produce it. Heterofermentative LAB are one of the best mannitol producers; these bacteria use the enzyme mannitol dehydrogenase (MDH) to convert fructose into mannitol with the concomitant oxidation of NAD(P)H. In this work, we describe the construction and characterization of MDH and lactate dehydrogenase (LDH) mutants from Fructobacillus tropaeoli CRL 2034. The draft genome of this strain, recently isolated from fig in our institute and selected for its excellent ability to produce mannitol from fructose, has been determined. A putative mdh gene in CRL 2034 was identified by in silico analysis; this gene has 1017 base pairs (bp) in length and encodes a protein of 338 amino acid (aa) residues. Downstream of mdh, a gene of 711 bp and encoding a putative permease protein of 236 aa was identified. Furthermore, three genes (named ldh1, ldh2 and ldh3) homologous to ldh genes from other organisms were identified in the genome of CRL 2034. Genes mdh, ldh1, ldh2 and ldh3 were independently deleted by single-crossover recombination in strain F. tropaeoli CRL 2034 by using the non-replicative delivery vector pRV300 and internal PCR-amplified fragments of each gene. The isolated erythromycin resistance mutants were submitted to PCR to confirm insertion, 16S rDNA sequencing and (GTG)5 Rep-PCR assays to corroborate that they were the same strain to the wild type. As expected, the mdh mutant strain did not display MDH activity while mannitol was not detected by HPLC; these results confirm the presence of a single mdh gene in F. tropaeoli CRL 2034. As MDH activity and mannitol production were not increased in the three ldh mutants, our results suggest that the different LDH activities are complementary or the existence of alternative ldh genes in the genome of strain CRL 2034. The construction of double and triple ldh deletion mutants, as well as complementation studies of the mdh mutant, are being carried out.