CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.

The α-glucosidase inhibitor acarbose, produced byActinoplanessp. SE50/110, is a well-known drug for the treatment of type 2 diabetes mellitus. However, the largely unexplored biosynthetic mechanism of this compound has impeded further titer improvement. Herein, we uncover that 1-epi-valienol and valienol, accumulated in the fermentation broth at a strikingly high molar ratio to acarbose, are shunt products that are not directly involved in acarbose biosynthesis. Additionally, we find that inefficient biosynthesis of the amino-deoxyhexose moiety plays a role in the formation of these shunt products. Therefore, strategies to minimize the flux to the shunt products and to maximize the supply of the amino-deoxyhexose moiety are implemented, which increase the acarbose titer by 1.2-fold to 7.4 g L⁻¹. This work provides insights into the biosynthesis of the C₇-cyclitol moiety and highlights the importance of assessing shunt product accumulation when seeking to improve the titer of microbial pharmaceutical products.

Polyketides serve as rich source of therapeutically relevant drug leads. The manipulation of polyketide synthases (PKSs) for generating derivatives with improved activities usually results in substantially reduced yields. Growing evidence suggests that type I PKS thioesterase (TE) domains are key bottlenecks in the biosynthesis of polyene antibiotics, such as pimaricin and amphotericin, and their unnatural derivatives. Herein, we elucidate the structure of the 26-membered macrolide-complexed TE domain from the pimaricin pathway (Pim TE), which specifies a spacious bifunnel-shaped substrate channel with a highly hydrophobic cleft proximal to the catalytic triad and a hydrophilic loop I region specific for the cyclization of amphiphilic polyene macrolide. Notably, the natural intermediate with C12-COOH is stabilized by a hydrogen-bond network, as well as by interactions between the polyene moiety and the hydrophobic cleft. Moreover, the bottleneck in processing the unnatural intermediate with C12-CH₃ is attributed to the unstable and mismatched docking of the curved substrate in the channel. Aided by an in vitro assay with a fully elongated linear polyene intermediate as the substrate, multiple strategies were adopted, herein, to engineer Pim TE, including introducing H-bond donors, enhancing hydrophobic interactions, and modifying the catalytic center. Efficient TE mutations with increased substrate conversion up to 39.2% in vitro were further conducted in vivo, with a titer increase as high as 37.1% for the less toxic decarboxylated pimaricin derivatives with C12-CH₃. Our work uncovers the mechanism of TE-catalyzed polyene macrolide formation and highlights TE domains as targets for PKS manipulation for titer increases in engineered unnatural polyketide derivatives.

In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.