Combinatorial Nonribosomal Peptide Synthetase Libraries Using the SEAM-Combi-OGAB Method

To overcome the difficulty of building large nonribosomal peptide synthetase (NRPS) gene cluster libraries, an efficient one-pot method using <i>Bacillus subtilis</i> was developed. This new method, named <u>S</u>eamed <u>E</u>xpress <u>A</u>ssembly <u>M</u>ethod (SEAM)-combi-<u>O</u>rdered <u>G</u>ene <u>A</u>ssembly in <i>Bacillus subtilis</i> (OGAB), combines the SEAM-OGAB approach for NRPS gene cluster construction with the combi-OGAB method for combinatorial DNA library construction to randomly swap DNA fragments for NRPS modules. In this study, NRPS gene clusters of plipastatin and gramicidin S were used as the starting material. The full length of each gene cluster was prepared as plasmid DNA by introducing restriction enzyme SfiI sites into the module border according to SEAM-OGAB. These two plasmids were mixed, digested with SfiI, ligated in a tandem repeat form, and used to transform <i>B. subtilis</i> according to the combi-OGAB method. While 64 of all the possible combinations were used in the calculation, 32 types of plasmid DNA were obtained from 50 randomly selected transformants. These transformants produced at least 30 types of peptides, including cyclic and linear variations with lengths ranging from 5 to 10 amino acids. Thus, this method enabled an efficient construction of NRPS gene cluster libraries with more than five module members, making it advantageous for applications in peptide libraries.