Anidulafungin, which noncompetitively inhibits β-(1,3)-D-glucan synthase in fungal cell wall biosynthesis, is the newest antifungal drug to be developed. Echinocandin B deacylase from Actinoplanes utahensis NRRL 12052 catalyzes the cleavage of the linoleoyl group of echinocandin B, a key step in the process of manufacturing anidulafungin. Unfortunately, the natural yield of echinocandin B nucleus is low. In our study, the echinocandin B deacylase gene was systematically overexpressed by genetic engineering in its original producer, A. utahensis, and in the heterologous hosts Streptomyces lividans TK24 and Streptomyces albus. The introduction of additional copies of the gene, under the control of PermE* or its native promoter, into hosts showed significant increases in its transcription level and in the efficiency of the bioconversion of echinocandin B to its nucleus. The conditions for the cultivation and bioconversion of A. utahensis have been optimized further to improve production. As a result, while the wild-type strain initially produced 0.36 g/liter, a concentration of 4.21 g/liter was obtained after the generation of a strain with additional copies of the gene and further optimization of the reaction conditions. These results are useful for enhancing echinocandin B nucleus production in A. utahensis. Our study could enable the engineering of commercially useful echinocandin B nucleus-overproducing stains.