Improvement of a d-biotin-hyperproducing recombinant strain of Serratia marcescens

We previously reported that a recombinant strain, SB412(pLGM304), was constructed from acidomycin-resistant mutants of Serratia marcescens and produced 200 mg of d-biotin per liter of a medium containing sucrose and urea (Sakurai et al., 1993a, b). In the present work, we intended to improve the d-biotin production. Both ethionine and S-2-aminoethylcysteine resistances were added to the host strain SB412, producing d-biotin at 20 mg l-1, and a resultant strain, ETA23, producing it at 33 mg l-1, was obtained. Cells of ETA23 did not maintain pLGM304 stably after greater than 30 generations under non-selective culture conditions. A new recombinant plasmid, pLGM304P, was constructed so as to be composed of pLGM304 and the parB locus, a plasmid-stabilizing element. ETA23 stably maintained pLGM304P after 50 generations under non-selective culture conditions. ETA23(pLGM304) produced 250 mg l-1 of d-biotin in a shaking flask under batch culture conditions and 500 mg l-1 in a jar fermentor under fed-batch culture conditions.