Metabolic engineering of lactic acid bacteria and characterization of novel enzymes for the production of industrially important compounds (2006)

Lactic acid bacteria (LAB) are a heterogeneous group of gram-positive bacteria that produce lactic acid as their main end-product during sugar fermentation. Because the LAB are able to rapidly lower pH through acid formation and additionally produce many flavor compounds, they are commonly used in the food and feed industry. LAB are also attractive organisms for metabolic engineering because their energy metabolism is generally not connected to their biosynthetic activity. Therefore, their sugar metabolism can be engineered without substantial interference to the biosynthesis pathways. This engineering ability has increased the interest in and significance of LAB as novel hosts for the production of valuable metabolites. \n\nDuring the past decade, tools for genetic modification of LAB have also been developed and considerable advancements in metabolic engineering of LAB have been made. For example, metabolic engineering of the pyruvate metabolism of LAB has resulted in efficient production of diacetyl and L-alanine. In addition, important advances have been made in the metabolic engineering of more complex biosynthetic pathways leading to products such as exopolysaccharides and vitamin B. \n\nIn this work, the main target was to modify the metabolism of LAB to produce industrially important compounds. Therefore, the first part of this work describes metabolic engineering to improve the production of mannitol, pyruvate and L-lactic acid in LAB. The second part describes isolation, cloning and characterization of genes encoding enzymes that are promising for the further development of genetically modified lactic acid bacteria. \n\nIn the first part, either the ldhD and ldhL genes, or ldhD alone, were inactivated using gene replacement techniques from a Lactobacillus fermentum strain known to be an efficient mannitol producer. With these gene inactivations two L. fermentum mutant strains were constructed, which produce mannitol and either pure L-lactate or pyruvate in a single process. A successful improvement of mannitol production by Leuconostoc pseudomesenteroides was also achieved by using random mutagenization to decrease its fructokinase activity. This would diminish the leakage of fructose from mannitol synthesis to the catabolic sugar pathway. Furthermore, the gene encoding the fructokinase and its putative promoter region were characterized. \n\nThe mannitol dehydrogenase (MDH) enzyme has a central role in producing mannitol from fructose in heterofermentative LAB. Therefore, in the second part of this work, the mannitol dehydrogenase gene (mdh) of Leuconostoc mesenteroides was isolated and overexpressed in E. coli to characterize its enzymatic properties. This was the first mdh gene characterized from heterofermentative LAB and it was later confirmed that mdh genes from this group of bacteria are clearly distinct from other bacterial mdh genes. Furthermore, we cloned a novel xylitol-4-dehydrogenase gene (xdh) of the gram-negative Pantoea ananatis. This is the first available sequence of a bacterial gene, which encodes an enzyme catalyzing the oxidation of xylitol to L-xylulose. This XDH enzyme offers an interesting opportunity to produce L-xylulose, for example, in recombinant LAB strains.