Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes found in bacteria and fungi, that synthesize a plethora of pharmaceutically relevant compounds. NRPSs consist of repeating sets of functional domains called modules, and each module is responsible for the incorporation of a single amino acid to the growing peptidyl intermediate. The synthetic logic of an NRPS resembles an assembly line, with growing biosynthesis intermediates covalently attached to the prosthetic 4'-phosphopantetheine (ppant) moieties of T (thiolation or transfer) domains for shuttling within and between modules. Therefore, NRPSs must have each T domain phosphopantetheinylated to be functional, and host organisms encode ppant transferases that affix ppant to T domains. Ppant transferases can be promiscuous with respect to the T domain substrate and with respect to chemical modifications of the ppant thiol, which has been a useful characteristic for study of megaenzymes and other systems. However, defined studies of multimodular megaenzymes, where different analogs are required to be affixed to different T domains within the same multimodular protein, are hindered by this promiscuity. Study of NRPS peptide bond formation, for which two T domains simultaneously deliver substrates to the condensation domain, is a prime example where one would want two T domains bearing different acyl/peptidyl groups. Here, we report a strategy where two NRPS modules that are normally part of the same protein are expressed as separate constructs, modified separately with different acyl-ppants, and then ligated together by sortase A of