Metabolic Engineering of Bacillus methanolicus for Efficient Production of Amino Acids from Methanol at Elevated Temperatures

A second major project is metabolic engineering of the restrictive, thermotolerant methylotroph Bacillus methanolicus for efficient production of L-lysine, glutamate, and other amino acids from methanol at 50°C. The demand for L-lysine, an essential amino acid in human, non-ruminant animal and fish nutrition, increases annually in proportion to global population growth. L-lysine is currently produced by microbial fermentation of carbohydrates at 30°C in excess of 500,000t/yr. In the future, additional carbon sources will be needed to meet increasing demand.

Microbes that grow at temperatures from 50°C to 65°C may be useful biocatalysts to efficiently manufacture amino acids and commodity chemicals from methanol because of the significant reduction in cooling very large reactors (400m³ to 1,000m³ liquid volumes).

Our group has developed molecular biology tools to metabolically engineer this unique halotolerant methylotrophic Bacillus. These methods include characterization of the major restriction and methylation systems, development of integrative and replicative plasmids, transformation methods, as well as identification of useful promoters and expression vectors. We investigate the pathways of methanol carbon assimilation and dissimilation using chemostats and small bioreactors with 13C NMR and isotope ratio mass spectroscopy. The regulation of carbon flux to aspartate or citrate and the biosynthesis of L-lysine or glutamate is being investigated in wild type isolates and mutant strains by PCR cloning of the genes which encode aspartokinase, citrate synthase, diaminopimelate decarboxylase, and other enzymes at key branch points of amino acid biosynthesis. The regulation of other genes involved in methanol assimilation is currently being investigated. Protein engineering methods are used to alter native enzyme structure and regulation to enhance the flux of L-lysine biosynthesis, to reduce in vivo formaldehyde toxicity, and to reduce the percentage of methanol carbon dissimilated to carbon dioxide.
Bacillus methanolicus may become the first high temperature microbial L-lysine manufacturing technology from a non-carbohydrate substrate.

Prof. Flickinger in Trondheim, Norway with collaborators from the Norweigan Technical University and SINTEF.

Current collaborators on this project include: (Left to right) T. Brautaset, S. Valla, A. Strøm, Ø. Jakobson, J. Kjell (National Technical Norweigan University), T.E. Ellingsen (SINTEFF, NTNU).

PDF of: BRIEF OVERVIEW of the CONVERSION of METHANOL to AMINO ACIDS by Bacillus methanolicus at 50 ºC