Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon

The E. coli biotin (bio) operon was modified to improve biotin production b y host cells: (a) the divergently transcribed wild-type bio operon was re-o rganized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ri bosomal binding site (RBS) was replaced with an artificial ass that resulte d in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin product ion, and bio gene expression with mini-cells and two-dimensional polyacryla mide gel electrophoresis, The modified E. coli bio operon was introduced in to a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material, Bio tin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth co nditions.