Promiscuous Enzymes Cooperate at the Substrate Level En Route to Lactazole A.

Enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) 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, 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 the 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. Our results reveal an unusual but well-defined assembly process where cyclodehydration, dehydroalanine formation, and azoline dehydrogenation events are intertwined due to minimal substrate recognition requirements characteristic of every lactazole enzyme. Additionally, each enzyme plays a role in directing LazBF-mediated dehydroalanine formation, which emerges as the central theme of the assembly process. Cyclodehydratase LazDE discriminates a single serine residue for azoline formation, leaving the remaining five as potential dehydratase substrates. Pyridine synthase LazC exerts kinetic control over LazBF to prevent the formation of overdehydrated thiopeptides, whereas the coupling of dehydrogenation to dehydroalanine installation impedes generation of underdehydrated products. Altogether, our results indicate that substrate-level cooperation between the biosynthetic enzymes maintains the integrity of lactazole assembly. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

Minimal lactazole scaffold for in vitro thiopeptide bioengineering.

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.

Identification of the common biosynthetic gene cluster for both antimicrobial streptoaminals and antifungal 5-alkyl-1,2,3,4-tetrahydroquinolines.

5-Alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) and streptoaminals (STAMs) are natural products isolated from the combined-culture of Streptomyces nigrescens HEK616 and Tsukamurella pulmonis TP-B0596. Despite their unique structures, their biosynthetic pathway has yet to be elucidated. In the present study, we conducted a feeding experiment using 13C-labeled acetates and demonstrated that 5aTHQs are likely synthesized by the action of polyketide synthase (PKS). Based on this observation, we identified the biosynthetic gene cluster for 5aTHQs. Interestingly, the same gene cluster was also responsible for the structurally-distinct STAMs. The gene cluster contains nine genes encoding one acyl carrier protein, two sets of ketosynthases (KSs) and chain length factors (CLFs), one aminotransferase/reductase bifunctional protein, two ketoreductases, and one thioesterase. KSs and CLFs are classified into the phylogenetically distinct clades from those of known type II PKSs. Heterologous expression of the biosynthetic genes and subsequent gene inactivation clearly indicated that all of the nine genes were required for the biosynthesis of both compounds. In the proposed biosynthetic pathway, chain elongation by PKS, reductive cleavage of a thioester bond, and subsequent transamination generate the core skeleton of both compounds. Differences in the oxidation states of the products result in a distinct cyclization mode to yield 5aTHQs and STAMs.

Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo.

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.