The first module of the assembly line, encoded by comK, contains a fatty-acyl AMP ligase (FAAL), a member of the adenylate forming enzymes. FAALs are often associated with NRPS/PKS pathways as a starter unit catalyzing the acylation of the first building block. In silico analysis revealed that all active sites in the Corramycin FAAL are present (see SI) and predicted a substrate specificity for long-chain fatty acids ranging from 5-12 C-atoms. Since no acylated Corramycin analogs were detected in the fermentation broth extracts, initially, we aimed to examine if the FAAL domain was indeed active and to investigate its substrate specificity. Therefore, we heterologously overexpressed the FAAL domain in E. coli BL21 (DE3) and performed a malachite green assay after protein purification. We observed activation of numerous linear fatty acids with different lengths by the FAAL domain (Supporting Figure ). The highest activity was observed for decanoic acid and the spectrum of activated fatty acid ceased at tridecylic acid and hexanoic acid. The broad substrate tolerance of the FAAL is not a surprising finding since lipopeptides often occur with various fatty acid chain lengths on the same peptide backbone. However, even upon re-inspection we did not find fatty acid-linked Corramycin derivatives in the extracts of the producer strains. There are numerous examples of lipopeptides harboring FAAL-incorporated fatty acid residues which are vital for their bioactivity. On the contrary, in the biosynthesis of the myxobacterial lipopeptide Vioprolide, a fatty acid-linked precursor is biosynthesized and the maturation of the active product is achieved by hydrolysis of the fatty acid. Since the Corramycin FAAL domain is evidently active, we speculated that inactive, acylated Corramycins are produced in terms of self-protection of the producer strain and, similarly to Vioprolide biosynthesis, subsequently hydrolyzed to their active form. However, in contrast to the Vioprolide case, we could thus far not identify any acylated Corramycin precursors and, consequently, further investigations are required in future experiments. A hypothetical pre-drug mechanism in Corramycin maturation could include ComB and ComC, a putative hydrolase and flippase-like exporter, respectively. The latter protein shows significant homology with glycosyl transferases. Both harbor an N-terminal peptide signal for periplasmic localization. The membrane-bound ComC might be involved in the export of putatively inactive, acylated Corramycin precursors to the periplasm, where hydrolysis by ComB potentially facilitates maturation to the final, active product. The second module in Corramycin biosynthesis, also encoded by comK, harbors an Fkbh-like domain belonging to the haloacid dehalogenase (HAD) superfamily. The same architecture of the first two modules (FAAL-ACP-C-HAD-T) was described for the Vioprolide assembly line, in which an unusual esterification process leads to the incorporation of a glycerate building block. However, Corramycins harbor an N-terminal C-4 butyric acid moiety and glycerate only contains three C-atoms. Thus, we assume that a glycerate moiety is incorporated and subsequently C-methylated in Corramycin biosynthesis. Based on the feeding of d3-methionine during fermentation of C. coralloides MCy10984 with subsequent analysis using LC-MS (see SI), we propose this reaction to be performed by ComJ, a radical-SAM methyltransferase. One intriguing chemical feature of Corramycins is the unprecedented -N-methyl-βhydroxy-histidine moiety. Based on the architecture of module 3 including a putative N-methyltransferase (N-MT) domain and an epimerization (E) domain, we assume that L-histidine is activated by the A domain, N-methylated by the N-MT domain and subsequently epimerized to -N-methyl-D-histidine by the E domain. Interestingly, to our knowledge no NRPS-associated N-MT domain acting on the side chain of an aromatic amino acid was described before. On the contrary, the biosynthetic pathway for the β-hydroxylation of histidine has been described. NikQ, a heme-protein, has been shown to facilitate the β-hydroxylation of histidine in Nikkomycin biosynthesis. Interestingly, free histidine is not accepted and has to be loaded onto a T domain by a respective A domain, before it is recognized by NikQ. However, we could not identify any cytochrome P450 dependent enzyme or other heme-containing proteins in the Corramycin BGC. Thus, we assume a different mechanism taking place in Corramycin biosynthesis that must be addressed in future experiments. Another unique feature of Corramycins is the unusual 5-amino-2,3-dihydroxy-4-methoxy valeroyl moiety. Based on the in silico analysis of modules 4 and 5 coupled with feeding experiments (Supplementary Table 3), we propose that aspartate is decarboxylated by ComE to β-alanine, which is incorporated by module 4, hydroxylated, methylated in cis by an O-methyltransferase domain, and finally extended by a C-2 body by the type I PKS module 5. Interestingly, two β-hydroxy-valines are found in Corramycins, which is a rarely observed building block in natural products. Independent supplementation of d8-L-valine, d6-L-hydroxy-valine and 13 C4- N-L-threonine during fermentation showed that only valine was accepted by the NRPS (Supplementary Figure S 22 and Table S 3). Consequently, the hydroxylation of this building block occurs either on-line or after product release. The β-hydroxy-valine moiety was also found in the myxobacterial nonribosomal peptide Myxoprincomide, however, its biosynthetic origin has thus far not been identified. Notably, no enzyme was identified in the Myxoprincomide BGC that catalyzes hydroxylation of valine, indicating that the responsible genes are located elsewhere in the genome of the producer strain. For Corramycin biosynthesis, further investigations are necessary to clarify the biosynthetic origin of β-hydroxy-valine, for example, by the independent deletion of genes encoding putative hydroxylases. Since the Corramycin production titer (< 0.1 mg L -1 ) by fermentation was low and the knowledge about optimal strain cultivation, production scale-up procedures as well as genetic manipulability of the natural producer were limited, we aimed to develop a total synthesis route. However, structure elucidation by NMR only revealed at that point the two-dimensional structure of Corramycin (Figure ). First, we performed an in silico analysis of the module and domain architecture of the Corramycin assembly line which allowed to predict the configuration of eight stereocenters (labelled with a star in Figure ). The HAD (FkbH) domain was proposed to load and dephosphorylate D-1,3bisphosphoglycerate and was experimentally shown to activate D-3-phosphoglycerate by the Vioprolide FkbH domain. Consequently, we proposed an R configuration for position 2 in Corramycin. The analysis of all C domain subtypes (see SI) revealed an L CL architecture for the C domain of module 3 (histidine) and a D CL architecture of the C domain of module 4.