Carrier proteins are central way stations in fatty acid, polyketide (PK) and nonribosomal peptide (NRP) biosynthesis. Precursors or intermediates of these natural products are covalently tethered to carrier proteins during chain elongation steps by phosphopantetheinyl moieties as thioesters, modified by catalytic partners in the assembly line, and subsequently transferred to downstream carrier proteins or released at the end of the assembly line. The two-module enterobaction synthetase was investigated as a model of nonribosomal peptide synthetases to understand the rules of recognition for carrier proteins. The growth of E. coli in minimal media supplemented with iron chelator 2-2' dipiridyl reported the production of enterobactin and the in vivo function of carrier protein mutants. We identified critical residues and surfaces on carrier proteins for domain communication by analyzing loss-of-function mutations; directed evolution of non-cognate carrier proteins yielded gain-of-function mutations for recognitions. These approaches collectively revealed insights about engineering or reprogramming nonribosomal peptide synthetases, for the goal of combinatorial biosynthesis. The region on carrier proteins for specific interactions with PPTases was subject to developing peptide tags for protein labeling. Phage display and selection yielded orthogonal peptide substrates for AcpS and Sfp for protein labeling in vitro or on cell surfaces. NMR studies on the AcpS substrate Al peptide revealed molecular details for the specific interactions between Al peptide and AcpS, and led to the development of eight-residue fragment A-4, which is an efficient peptide tag for labeling.