The ever-growing drug resistance problem worldwide highlights the urgency to discover and develop new drugs. Microbial natural products are a prolific source of drugs. Genome sequencing has revealed a tremendous amount of uncharacterized natural product biosynthetic gene clusters (BGCs) encoded within microbial genomes, most of which are cryptic or express at very low levels under standard culture conditions. Therefore, developing effective strategies to awaken these cryptic BGCs is of great interest for natural product discovery. In this study, we designed and validated a Transcription–Translation in One (TTO) approach for activation of cryptic BGCs. This approach aims to alter the metabolite profiles of target strains by directly overexpressing exogenous rpsL (encoding ribosomal protein S12) and rpoB (encoding RNA polymerase β subunit) genes containing beneficial mutations for natural product production using a plug-and-play plasmid system. As a result, this approach bypasses the tedious screening work and overcomes the false positive problem in the traditional ribosome engineering approach. In this work, the TTO approach was successfully applied to activating cryptic BGCs in three Streptomyces strains, leading to the discovery of two aromatic polyketide antibiotics, piloquinone and homopiloquinone. We further identified a single BGC responsible for the biosynthesis of both piloquinone and homopiloquinone, which features an unusual starter unit incorporation step. This powerful strategy can be further exploited for BGC activation in strains even beyond streptomycetes, thus facilitating natural product discovery research in the future.
Abstract The biosynthetic investigations of microbial natural products continuously provide powerful biocatalysts for the preparation of valuable chemicals. Practical methods for preparing ( S )‐3‐aminopiperidine‐2,6‐dione ( 2 ), the pharmacophore of thalidomide ( 1 ) and its analog drugs, are highly desired. To develop a biocatalyst for producing ( S )‐ 2 , we dissected the domain functions of IdgS, which is responsible for the biosynthesis of indigoidine ( 3 ), a microbial blue pigment that consists of two 2 ‐like moieties. Our data supported that the L‐glutamine tethered to the indigoidine assembly line is first offloaded and cyclized by the thioesterase domain to form ( S )‐ 2 , which is then dehydrogenated by the oxidation (Ox) domain and finally dimerized to yield 3 . Based on this, we developed an IdgS‐derived enzyme biocatalyst, IdgS‐Ox* R539A, for preparing enantiomerically pure ( S )‐ 2 . As a proof of concept, one‐pot chemoenzymatic synthesis of 1 was achieved by combining the biocatalytic and chemical approaches.
Jadomycin production is under complex regulation in Streptomyces venezuelae. Here, another cluster-situated regulator, JadR*, was shown to negatively regulate jadomycin biosynthesis by binding to four upstream regions of jadY, jadR1, jadI and jadE in jad gene cluster respectively. The transcriptional levels of four target genes of JadR* increased significantly in ΔjadR*, confirming that these genes were directly repressed by JadR*. Jadomycin B (JdB) and its biosynthetic intermediates 2,3-dehydro-UWM6 (DHU), dehydrorabelomycin (DHR) and jadomycin A (JdA) modulated the DNA-binding activities of JadR* on the jadY promoter, with DHR giving the strongest dissociation effects. Direct interactions between JadR* and these ligands were further demonstrated by surface plasmon resonance, which showed that DHR has the highest affinity for JadR*. However, only DHU and DHR could induce the expression of jadY and jadR* in vivo. JadY is the FMN/FAD reductase supplying cofactors FMNH₂/FADH₂ for JadG, an oxygenase, that catalyses the conversion of DHR to JdA. Therefore, our results revealed that JadR* and early pathway intermediates, particularly DHR, regulate cofactor supply by a convincing case of a feed-forward mechanism. Such delicate regulation of expression of jadY could ensure a timely supply of cofactors FMNH₂/FADH₂ for jadomycin biosynthesis, and avoid unnecessary consumption of NAD(P)H.
A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.
Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .
We confirm the previously revised stereochemistry of spiroviolene by X-ray crystallographically characterizing a hydrazone derivative of 9-oxo-spiroviolane, which is synthesized from hydroboration-oxidation of spiroviolene followed by oxidation of the resultant hydroxy group. An unexpected thermal boron migration occurred during the hydroboration process of spiroviolene, that have resulted in the production of a mixture of 1α-hydroxy-spiroviolane, 9α- and 9β-hydroxy-spiroviolane after oxidation. The assertion of the cis-oriented 19- and 20-methyl groups has provided further support for the revised cyclization mechanism of spiroviolene.
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