Gene editing enables rapid engineering of complex antibiotic assembly lines.

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Reconstitution of a Highly Reducing Type II PKS System Reveals 6π-Electrocyclization Is Required for o-Dialkylbenzene Biosynthesis.

Natural products containing an o-dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the o-dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds 1 and 2 in Streptomyces sp. SANK 60404, which contains a rare o-dialkylbenzene moiety, and successfully reconstituted the biosynthesis of 1 using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the o-tolyl group within the o-dialkylbenzene moiety, where the triene intermediate 3 loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C2-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6Z configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an o-dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form o-dialkylbenzenes in nature.

Discovery of an Antibacterial Isoindolinone-Containing Tetracyclic Polyketide by Cryptic Gene Activation and Characterization of Its Biosynthetic Gene Cluster.

The major setback in natural product screening is the decreasing hit rate of novel bioactive compounds containing new chemical skeletons. Here we report the identification and biosynthesis of isoindolinomycin (Idm), an unprecedented bioactive polyketide with a novel isoindolinone-containing tetracyclic skeleton. Idm was discovered through the screening of rifampicin-resistant ( rif) mutants that were generated from nine actinomycete strains used in this study. Of the 114 rif mutants isolated, the mutant S55-50-5 was found to overproduce Idm, which is almost undetectable in the wild-type Streptomyces sp. SoC090715LN-16. An in silico analysis coupled with gene deletion experiments revealed a biosynthetic idmB gene cluster that is responsible for the production of Idm. The biosynthetic studies of Idm primarily focused on the formation of the five-membered ring in the tetracyclic structure and the attachment of the methyl group to the core structure. In addition, a malachite green phosphate assay performed using a stand-alone adenylation domain ( idmB21) demonstrated the involvement of glycine in the formation of the isoindolinone-containing skeleton. This study contributes to an increase in the structural diversity of polyketides and paves the way toward an understanding of the complete biosynthetic pathway of a novel class of tetracyclic polyketides.

Methylbenzene-Containing Polyketides from a Streptomyces that Spontaneously Acquired Rifampicin Resistance: Structural Elucidation and Biosynthesis.

Conventional screening for novel bioactive compounds in actinomycetes often results in the rediscovery of known compounds. In contrast, recent genome sequencing revealed that most of the predicted gene clusters for secondary metabolisms are not expressed under standard cultivation conditions. To explore the potential metabolites produced by these gene clusters, we implemented a cryptic gene activation strategy by screening mutants that acquire resistance to rifampicin. The induction of rifampicin resistance in 11 actinomycete strains generated 164 rifampicin-resistant mutants (rif mutants). The comparison of the metabolic profiles between the rif mutants and their wild-type strains indicated that one mutant (TW-R50-13) overproduced an unidentified metabolite (1). During the isolation and structural elucidation of metabolite 1, an additional metabolite was found; both are unprecedented compounds featuring a C5N unit and a methylbenzene moiety. Of these partial structures, the biosynthesis of the latter has not been reported. A feeding experiment using (13)C-labeled precursors demonstrated that the methylbenzene moiety is most likely synthesized by the action of polyketide synthase. The gene deletion experiments revealed that the genes for the methylbenzene moiety are located at a different locus than the genes for the C5N unit.