Using biomass support particles (BSPs) as a cell immobilized matrix, immobilized recombinant Streptomyces lividans continuously produced phospholipase D (PLD) in a yield of about 1.5 x 10(4) U/L in each of eight batches. In contrast to the original strain Streptoverticillium cinnamoneum, this heterologous expression system with an immobilization method is capable of producing secretory PLD with an 8-fold greater efficiency. The presence of both glucose and tryptone in the initial culture medium also promoted secretory production, and PLD activity around 3.0 x 10(4) U/L were achieved. In addition, the promoter region of PLD ORF was deduced, and three types of plasmid having different lengths of promoter sequence were constructed. The deduced sequence had same effect on either of PLD production or mycelium immobilization, and the transformants harboring each of three plasmids showed the similar cultivation profiles (3.0 x 10(4) U/L). A combination of the immobilization method with BSPs and S. lividans transformant harboring the deduced plasmid has the potential for producing secretory PLD in the culture supernatant continuously. (c) 2007 Elsevier Inc. All rights reserved.