Background: Activation of silent biosynthetic gene clusters (BGCs) in marine-derived actinomycete strains is a feasible strategy to discover bioactive natural products. Actinoalloteichus sp. AHMU CJ021, isolated from the seashore, was shown to contain an intact but silent caerulomycin A (CRM A) BGC-cam in its genome. Thus, a genome mining work was preformed to activate the strain’s production of CRM A, an immunosuppressive drug lead with diverse bioactivities. Results: To well activate the expression of cam, ribosome engineering was adopted to treat the wild type Actinoalloteichus sp. AHMU CJ021. The initial mutant strain XC-11G with gentamycin resistance and CRM A production titer of 42.51 ± 4.22 mg/L was selected from all generated mutant strains by gene expression comparison of the essential biosynthetic gene-camE. The titer of CRM A production was then improved by two strain breeding methods via UV mutagenesis and cofactor engineering-directed increase of intracellular riboflavin, which finally generated the optimal mutant strain XC-11GUR with a CRM A production titer of 113.91 ± 7.58 mg/L. Subsequently, this titer of strain XC-11GUR was improved to 618.61 ± 16.29 mg/L through medium optimization together with further adjustment derived from response surface methodology. In terms of this 14.6 folds increase in the titer of CRM A compared to the initial value, strain XC-GUR could be a well alternative strain for CRM A development. Conclusions: Our results had constructed an ideal CRM A producer. More importantly, our efforts also had demonstrated the effectiveness of abovementioned combinatorial strategies, which is applicable to the genome mining of bioactive natural products from abundant actinomycetes strains.

Acute kidney injury (AKI) refers to clinical syndromes culminating in rapidly reduced renal function associated with inflammation and the demise of renal tubular epithelial cells. Current research aims to develop strategies which prevent tubular cell death. Here, based on the involvement of histone deacetylases (HDACs) in renal physiology and their established role in renal fibrosis, we investigated the mechanistic contributions of HDACs using a mouse model together with in vitro studies employing human renal epithelial cells. We found HDAC3 expression was upregulated in mouse renal tubules after ischemia/reperfusion and cisplatin treatment. Instructively, treatment with the HDAC3 selective inhibitor RGFP966 exerted potent protective effects, attenuates acute kidney injury in both in vivo and in vitro models. Moreover, RGFP966 was found to reduce inflammation and injury caused by cisplatin and hypoxia-reoxygenation in HK2 cells with transcriptome sequencing revealing that RGFP966 significantly inhibited the upregulation of the necroptosis initiator, RIPK1. Cellular thermal displacement assay and molecular docking demonstrated the physical binding of RGFP966 to HDCA3. In addition, RIPK1 knockdown cell assay signified that RGFP966 targeted RIPK1 and inhibited RIPK1 kinase activity. In summary, these findings established the efficacy of the HDAC3 inhibitor RGFP966 in treating AKI.

Pictet-Spengler (PS) reactions are pivotal in the biosynthesis of bioactive alkaloids and pharmaceuticals, yet key structural details underlying their enzymatic catalysis remain insufficiently understood. We identified AsKslB from Actinosynnema sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of l-tryptophan (l-Trp) and α-ketoglutarate (α-KG) to form kitasetalic acid (KA), a tetrahydro-β-carboline (THβC). High-resolution crystal structures of apo, substrate-, intermediate-, and product-bound forms elucidate the full catalytic trajectory and key residues. Crucially, the elusive iminium ion intermediate (IM-1) and a synchronously released water molecule are captured, providing direct structural evidence for the initiating cyclization step of Pictet-Spengler reaction. Glu276 undergoes conformational changes essential for catalysis. These findings offer detailed mechanistic insights into Pictet-Spenglerase function and establish AsKslB as a promising biocatalyst for stereoselective N-heterocycle synthesis.