In the present study, we sought to confirm a putative non-ribosomal peptide synthase (NRPS) - polyketide synthase (PKS) gene cluster in marine sponge-associated bacterium with cytotoxic activity and elucidate the gene's structural information. The genomic library of the marine sponge Hymeniacidon perleve-associated bacterium Pseudoalteromonas sp. strain NJ631 was constructed using a pCC1FOS fosmid. Positive clones that covered the whole gene cluster region of hybrid NRPS-PKS were selected for shotgun sequencing. The results obtained from BlastX and open reading frame (ORF) analysis indicated that there are 3 big ORFs, NJA1, NJA2, and NJA3, that encoded proteins with similarities to amino acid adenylation, beta-ketoacyl synthase, and non-ribosomal peptide synthase, respectively, from different organisms. The results gave us a clue that there could be PKS or NRPS modules in the 3 ORFs. Further analysis demonstrated 3 ORFs encoding 2 NRPS modules, 1 PKS module, and 3 NRPS modules. Using the specificity-conferring selection rule, the substrate specificity of 4 adenylation (A) domains (A2, A3, A4, and A5) were successfully predicted, and the amino acids of the substrate specificity were glutamic acid - glutamine, serine, D-serine, and Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid), respectively.