Technical Abstract: Gram-positive bacteria have been explored to convert lignocellulosic biomass to biofuel and bioproducts. Our long term 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 is now considered a model for genetic manipulations of LAB. In this study, L. plantarum TF103 strain, in which two of the chromosomal L-ldh and D-ldh genes are inactivated, was used to introduce additional mutations on the chromosome to eliminate undesired fermentation products. We targeted the acetolactate synthase gene (als) that converts pyruvate to acetolactate, to eliminate the production of acetoin and 2, 3-butanodial. A small portion of the als coding region was deleted, with an erythromycin resistance gene inserted at the deletion site. This pBluescript derivative was then directly introduced into L. plantarum TF103 cells to create mutations under selection pressure. The resulting erythromycin resistant (Em**r) 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 thus generated via targeted homologous recombination using a Gram-negative cloning vector, eliminating the use of a shuttle vector. This method should facilitate research in targeted inactivation of other genes in lactic acid bacteria.