To maintain the pET28-derived plasmids, the media was supplemented with kanamycin (50 μg/ml) throughout the expression cultures. When appropriate, the media was further supplemented with chloroamphenicol (20 μg/ml) to maintain the BirA containing pACYC184 plasmid. E. coli BL21(DE3) cells transformed with appropriate plasmids were grown for 5 h at 30°C, and a 10-ml sample adjusted to OD (600) =1 was then transferred to a 2-l fedbatch fermentor (LabFors®). To induce protein expression, IPTG (1 mM) was added at OD (600) ∼25 and the culture was continued for an additional 3 h at 42°C (for in vivo biotinylation of the product, the induction media was further supplemented with biotin (Sigma #B4501, 125 μg/ ml)). Samples were analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before and after IPTG induction. At the end of the induction culture, protease inhibitor (PMSF, 80 μg/l) was added, and cells were lysed in a cell disrupter (Constant Cell Disruptor Systems set at 2,300 bar) and the released inclusion bodies were isolated by centrifugation (Sorval RC6, 20 min, 17,000×g). The inclusion bodies were washed twice in PBS, 0.5% NP-40 (Sigma), and 0.1% deoxycholic acid (Sigma) and extracted into urea-Tris buffer (8 M urea, 25 mM Tris, pH 8.0), and any contaminating DNA was precipitated with streptomycin sulfate (1% w/v).