Isolation and characterization of a ColE1 plasmid containing the entire Bio gene cluster of Escherichia coli K12

The effect of the overexpression of the bioABFCD operon on the biotin biosynthetic pathway was investigated in an Escherichia coli K12 bioR mutant B ioC,Bi oH with a chromosomal deletion for the biotin operon. When transformed with a multicopy number plasmid NH2 J containing bioABFCD, this strain synthesized 10,000CH~-CH-C00H + times more biotin than a wild-type E. coli strain. In or-BioF der to further increase biotin production, the bioA and bioB operons were subcloned into plasmids with stronger promoters and in some cases optimal ribosome binding sites. The new constructions led to the accumulation of large amounts of soluble Bio proteins (although not BioA BioC) but did not improve biotin production. In all the constructed strains, BioA, BioD, and BioB activities were greatly amplified but these activities did not correlate with the level of protein synthesis. These strains ac-B i o O cumulated only low levels of vitamers, suggesting that the major limiting step for higher biotin production occurs upstream from the first intermediate of the Bio pathway we assayed (7,keto-8-aminopelargonic acid). As BioC overproduction was shown to impair cell growth, we could not determine if this early step of the pathway was limiting.

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