Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides.

Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the ‘flexible' in vitro translation system, referred to as the FIT–GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo . The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease.

Enzymes involved in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, and yet usually, the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide.Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level, and supplement this study with substrate scope analysis for participating enzymes. Combined, our results reveal a dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined enzymatic action, and the overall substrate-level cooperation between the enzymes. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering..

18Enzymes involved in ribosomally synthesized and post-translationally modified peptide 19 (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse 20 substrates. When several such enzymes act together during precursor peptide maturation, 21 a multitude of products can form, and yet usually, the biosynthesis converges on a single 22 natural product. For the most part, the mechanisms controlling the integrity of RiPP 23 assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model 24 thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. 25Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of 26 biosynthetic events at the individual modification level, and supplement this study with 27 substrate scope analysis for participating enzymes. Combined, our results reveal a 28 dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined 29 enzymatic action, and the overall substrate-level cooperation between the enzymes. This 30 work advances our understanding of RiPP biosynthesis processes and facilitates 31 thiopeptide bioengineering. 32 3 Main text 33 Ribosomally synthesized and post-translationally modified peptides (RiPPs) are 34 structurally and functionally diverse natural products united by a common biosynthetic 35 logic. 1 Usually during RiPP maturation, biosynthetic enzymes utilize the N-terminal 36 sequence of a ribosomally produced precursor peptide as a recognition motif (leader 37 peptide; LP) and install post-translational modifications (PTMs) in the C-terminal section of 38 the same substrate (core peptide; CP). This mode of action leads to relaxed substrate 39 requirements around the modification sites, which is often exemplified by one RiPP 40 enzyme introducing multiple PTMs in a single substrate. In one extreme case, a single 41 enzyme epimerizes 18 out of 49 amino acids in polytheonamide A precursor peptide 42 during its biosynthesis. 2,3 Unique enzymology of RiPP biosynthetic enzymes has come 43 under intense scrutiny in the recent years, which explained observed substrate specificities 44 in many cases. 4,5,14,15,6-13 During biosynthesis of complex RiPPs, when multiple enzymes 45 capable of differentially modifying their substrate act together, a multitude of products can 46 often form, and yet usually the biosynthetic pathway manages to produce a single natural 47

The new subfamily of type II PKS gene cluster is responsible for biosynthesis of structurally distinct streptoaminals (STAMs) and 5-alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs).