ARS | Publication request: Chromosomal Inactivation of the Als Gene in Lactobacillus Plantarum Strain Tf103 to Eliminate Undesired Fermentation Products

Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract
Publication Acceptance Date: August 3, 2006
Publication Date: August 3, 2006
Citation: Liu, S. 2006. Chromosomal inactivation of the als gene in Lactobacillus plantarum strain TF103 to eliminate undesired fermentation products [abstract]. Society of Industrial Microbiology. p. 166.

Technical Abstract: Gram-positive bacteria have been explored to convert lignocellulosic biomass to biofuel and bioproducts. Our ultimate goal is to create genetically engineered lactic acid bacteria (LAB) strains that convert agricultural biomass into ethanol and other value-added products. The immediate approaches toward this goal involve genetic manipulations by either introducing ethanol production pathway genes or inactivating pathways genes that lead to production of undesired byproducts. The widely studied species Lactobacillus plantarum has been used as a model for genetic manipulations of LAB. In this study, we used L. plantarum strain TF103, in which the chromosomal L-ldh and D-ldh genes had been inactivated, to generate a third chromosomal mutation of the als gene that encodes acetolactate synthase. Acetolactate synthase converts pyruvate to acetolactate, which is the intermediate for synthesis of acetoin and 2,3-butanodial. In strain TF103, when lactate production was reduced significantly, acetoin and 2,3-butanodial were increased along with increased ethanol production. Therefore, if the als gene is inactivated in TF103, cellular pyruvate might be redirected toward more ethanol. A pBluescript derivative plasmid was constructed in which a small portion of the als coding region was deleted, with an erythromycin resistance gene inserted at the deletion site. This plasmid was directly introduced into L. plantarum TF103 cells to create mutations under selection pressure. The resulting erythromycin resistant (Emr) TF103 strain appears to have chromosomal mutations of both the als and the adjacent lysP genes as revealed by PCR and Southern blot analyses. Mutations were generated via targeted homologous recombination using a suicidal cloning vector, eliminating the use of a shuttle vector. This method might be applicable to targeted inactivation of other genes in other species of lactic acid bacteria.