Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 8/25/2007
Publication Date: 8/30/2008
Citation: Hilker, B.L., Fukushige, H., Hou, C.T., Hildebrand, D. 2008. Some properties of a self-sufficient cytochrome P-450 monooxygenase system from Bacillus megaterium strain ALA2. In: Hou, C.T., editor. Biocatalysis and Bioenergy. New York, NY:John Wiley & Sons. p. 291-308 Interpretive Summary: USDA patented microbial culture Bacillus megaterium ALA2 produced many new oxygenated fatty acids with high potential in industrial applications. We reviewed literature on the biocatalyst (monooxygenase enzyme) of Bacillus megaterium and related species. We identified the initial enzyme of strain ALA2 as a catalytically self-sufficient cytochrome P450 monooxygenase, similar to many well-studied Bacillus enzymes. The gene of this enzyme was isolated and characterized. This enzyme is relatively stable and is good for industrial applications. The isolated monooxygenase gene can be transferred into other microbes such as Escherichia coli or into crop plants to produce desired products. Success in the transferring genes to microbes or crop plants can increase productivity of desired products and benefit both U.S. farmers and the public.
Technical Abstract: We reviewed the many interesting and related in vivo products derived from reactions of the B. megaterium strain ALA2 and various related polyunsaturated fatty acids (PUFA) substrates. Products obtained from the omega-6 PUFAs (linoleic acid, gamma-linolenic acid, and arachidonic acid) possessed diepoxy bicyclic structures, tetrahydrofuranyl rings, and/or trihydroxy groups in their molecules while the products from omega-3 PUFAs (alpha-linolenic acid, EPA, and DHA) only held hydroxyl THFAs. All of these products were similar enough to likely have resulted from parallel oxidation mechanisms. Despite the chemical structure of products produced by strain ALA2 depending heavily on the type of PUFA substrate employed, all of the chemistry in each PUFA class was identical when viewed from the omega terminal end of the substrates. Even the omega-6 to omega-3 reaction comparisons showed similar highest numbered double bond hydration, hydroxylations, and/or dehydration-based ring closures. Also reported is the initial gene sequence data for a cloned strain ALA2-based P450 enzyme(s). As suspected from its types of in vivo chemistries, this B. megaterium strain included a monooxygenase enzyme of the soluble, self-sufficient P450BM-3 sub-class that was highly homologous with the wild type protein. Not surprisingly, this new enzyme also possessed a significant high homology in all of the expected reductase regions as well. The in vitro reactions and substrate-binding characteristics of this enzyme will be further characterized in the near future.