For the first approach we identified accessible myxovalargin producers through a search across 2,500 LC-MS datasets from a previous secondary metabolome study . Among the small number of candidates, strain MCy6431 -by 16S rRNA sequence comparison likely a species of the genus Pyxidicoccus -showed more favorable production and growth characteristics when compared to the literature-reported producer Mx f65 and was also amenable to genetic manipulation. Strain MCy6431 was thus subjected to media optimization and up-scaling of fermentation to facilitate myxovalargin production (see SI). Furthermore, its 13.2 Mbp genome sequence was determined to enable elucidation of the biosynthetic pathway for myxovalargin. The myxovalargin scaffold comprises mainly non-proteinogenic amino acid building blocks including several D-valine, D-alanine, and D-arginine as well as (S)-beta-tyrosine, dehydro-valine, dehydro-isoleucine and hydroxy-valine moieties and was therefore assumed as the product of a nonribosomal peptide synthetase (NRPS)-type biosynthesis route . Targeted gene inactivation connected the compound class to a large biosynthetic gene cluster (BGC) spanning some 70 kbps and comprising at least two operons, with core biosynthesis functions encoded by genes mxvA-E. The number and type of domains encoded within the mxv biosynthetic locus as well as the biochemical characteristics of individual domains predicted through detailed bioinformatic analysis largely agree with a model for NRPS-directed biosynthesis of myxovalargins as shown in Fig. (see also SI). A tyrosine 2,3-aminomutase supplying the striking (S)-beta-tyrosine building block is encoded by mxvJ, which is a homolog of the aminomutase-encoding gene characterized from Myxococcus in an earlier report , and its role in Myx biosynthesis was confirmed by targeted gene inactivation and complementation through supplementation of b-tyrosine (SI). Since the Myx assembly line does not employ a type-1 thioesterase (mxvF instead encodes a type-2 thioesterase with proofreading function only), Myx biosynthesis intriguingly terminates with a Cdomain catalyzed incorporation of agmatine, as underpinned by our feeding study (see SI). The finding and positioning of D-configured amino acids in Myxs agrees widely with the presence of integral epimerase (E) domains encoded on mxvA,C,D,E as well as with the respective downstream C-domain classification into LCL or DCL types , however, with two remarkable exceptions: Module 7 contains an E domain, which suggests D-valine at the respective position in the peptide, while module 10 lacks an E domain, which is contrary to the D-valine assigned to this position in the published structure of MyxA . This stereochemical discrepancy could not be rationalized in light of the assumed colinear NRPS-based architecture of Myx biosynthesis and therefore prompted us to first verify the genetic information, which was performed by re-sequencing and southern blotting (SI). Our observations ultimately led us to challenge the original stereochemical assignment at these positions. Consequently, we carried out an independent determination of Myx configuration using a precursor feeding approach coupled with mass spectrometry (see SI). The outcome of our MS/MS-based analysis of labelled fragment ions (cp. Fig. ) unambiguously confirmed the altered stereochemical assignment of myxovalargin building blocks as D-valine incorporated by module 7 and L-valine for module 10. We thus show the revised configuration throughout this article and the structural revision also informed the total synthesis approach.