The bacterium Bacillus methanolicus can utilize methanol as the sole carbon and energy source. This trait, together with a relative high optimum growth temperature, makes this organism a highly promising biocatalyst for conversion of methanol into commercially useful compounds such as the amino acids L-lysine and L-glutamate. Compared to sugars, methanol is regarded an interesting alternative raw material for microbial bioprocesses. Through a metabolic engineering approach, we characterize and improve the metabolic routes and enzymes involved in methanol uptake and assimilation, in addition to biosynthesis and export of L-lysine and L-glutamate in this organism. During this work the genetic and regulatory basis for B. methanolicus methylotrophy was unraveled. Critical methylotrophy genes are encoded by a 19 kb large multicopy natural plasmid and there is a concerted transcriptional co-regulation of chromosomal and plasmid encoded methylotrophy genes. Based on this knowledge we have generated recombinant B. methanolicus strains with significantly improved specific growth rate on methanol. Recently, several amino acid biosynthetic pathway genes have been cloned, and their individual impact on production of L-lysine and L-glutamate is now being extensively investigated. In particular, manipulations with the three aspartokinase isozymes resulted in up to 60-fold improved L-lysine production without negatively affecting the specific growth rate of the cells. Moreover, co-manipulation with multiple enzymes has addititive effects and results in further improved L-lysine production levels.