Genetic manipulation in Lactobacillus brevis for butanol synthesis

Abstract

Gram-positive bacteria are well known for the potential of producing antimicrobial compounds, metabolites and other value added products. Among the gram-positive bacteria, lactic acid bacteria especially, Lactobacilli have gained attention nowadays, due to the production of industrial important biochemical, antimicrobial peptides, and biofuels. Naturally associated characteristics such as wide range of substrate utilization, availability of genomic information, small genome size and tolerance to low pH and high alcohol concentration with Lactobacillus makes them ideal strain for biofuel production. This work addresses the characteristics of metabolites synthesized by the Lactobacillus with particular biofuels (biobutanol). In addition, the genetic and metabolic engineering approaches used for improved production of butanol. Strategies to overcome the drawback of metabolic engineering (limited supply of cofactors on cloning of external pathway or gene and product toxicity) are also performed. The vector pHYCAT has the heterologous CAT gene promoter while pHYCYC have homologous promoter which is used as the selectable marker for Lactobacillus brevis. These vectors have high efficiency in E. coli but low in Lactobacillus. Chemical mutagenesis with ethyl methansulphate (EMS) was used to develop mutants of the Lactobacillus brevis ATCC367 that have downregulated lactate dehydrogenase activity. Three mutants (M-43, M-135 and M-200) showed 1, 1.6 and 4 fold reduction in LDH activity as compared to the wild type Lactobacillus brevis but growth rate and substrate utilization were unaltered as compared to the control. Lactate dehydrogenase from mutant M-200 which had 4 fold decreases in enzymatic activity was purified to homogeneity with a molecular size of 35KDa, maximum activity of 0.006IU at pH-7.0 and temperature 250C. The generated mutants were further used for the production of the butanol. The genes will be transferred with the constructed vector.