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Genetic engineering of Bacillus subtilis for the commercial production of riboflavin. Journal of Industrial Microbiology & Biotechnology 22, 8-18.
| Perkins J.B., Sloma, A., Hermann, T., Theriault, K., Zachgo, E., Erdenberger, T., Hannett, N., Chatterjee, N. P., Williams, V., II, Rufo, G. A., Hatch, R., Pero, J. (1999).
| Recombinant DNA engineering was combined with mutant selection and fermentation improvement to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic operon (rib operon) integrated at two different sites in the B. subtilis chromosome. The modified rib operons are expressed constitutively from strong phage promoters located at the 5' end and in an internal region of the operon. The engineered strain also contains purine analog-resistant mutations designed to deregulate the purine pathway (GTP is the precursor for riboflavin), and a riboflavin analog-resistant mutation in ribC that deregulates the riboflavin biosynthetic pathway.
Riboflavin (vitamin B2) is an essential vitamin; a precursor to the coenzymes flavin mononucleotide (FMN) and flavine adenine dinucleotide (FAD), which is synthesized by plants and microorganisms, but not by higher organisms. Recombinant DNA engineering, combined with mutant selectionand fermentation improvement was used to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The modified riboflavin biosynthetic operon (rib operon) was introduced in multiple copies into the bacterial chromosome. Expression was increased by mutating a regulatory gene (ribC), by replacing/bypassing two different promoter/regulatory sites with strong, constitutive promoters, and by increasing the copy number of the genes.
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