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Methylketones are broadly distributed in nature and perform a variety of functions. Most microorganisms are thought to produce methylketone by abortive β-oxidation of fatty acid catalytic metabolism. However, two methylketone synthetase genes in wild tomatoes are reported to synthesize methylketone using intermediates of the fatty acids biosynthetic pathway. In our previous study on Trojan horse-like interactions between the bacterium Bacillus nematocida B16 and its host worm, the chemical 2-heptanone was found to be an important attractant for the hosts. So here we used this model to investigate the genes involved in synthesizing 2-heptanone in microorganisms. We identified a novel methylketone synthase gene yneP in B. nematocida B16 and found enhancement of de novo fatty acid synthesis during 2-heptanone production. Interestingly, a homolog of yneP’ existed in the non-pathogenic species Bacillus subtilis 168, a close relative of B. nematocida B16 that was unable to lure worms, but GC-MS assay showed no 2-heptanone production. However, overexpression of yneP’ from B. subtilis in both heterologous and homologous systems demonstrated that it was not a pseudogene. The transcriptional analysis between those two genes had few differences under the same conditions. It was further shown that the failure to detect 2-heptanone in B. subtilis 168 was at least partly due to its conversion into 6-methyl-2-heptanone by methylation. Our study revealed methylketone biosynthesis of Bacillus species, and provided a co-evolution paradigm of second metabolites during the interactions between pathogenic/non-pathogenic bacteria and host.

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Active natural products are an important source of drug discovery due to their unique biocompatibility, novel-structural framework and extensive pharmacological activities. The target identification of natural products is the key to thoroughly understanding their mechanism of action and guiding their subsequent structure optimization. Lathyrane diterpenoids isolated from the seeds of Euphorbia lathyris in our laboratory have been proved to possess good anti-inflammatory activities in lipopolysaccharide (LPS)-induced macrophages, but their precise target and mechanism of action remain unclear. Herein, we employed PROTAC technology combined with quantitative proteomic analysis to identify the potential targets of lathyrane diterpenoids in macrophages. ZCY-PROTAC, synthesized based on Lathyrol, the core scaffold structure of the most active natural compound (2S,3S,4S,5R,9S,11R,15R)-15-acetoxy-3- cinnamoyloxy-5-hydroxy-14-oxolathyra-6(17),12E-diene, ZCY-001), intensively degraded a target protein MAFF in mouse and human cells. MST, CETSA and DARTS assays confirmed the direct binding of MAFF with Lathyrol or ZCY020, a natural product sharing the same core scaffold structure. Further mechanism studies showed that ZCY020 was capable of inhibiting the formation of a MAFF homodimer, promoting MAFF-Nrf2 heterodimerization, and thus regulating the transcription and expression of downstream protein HO-1, thereby exerting antioxidant, anti-inflammatory activity and promoting protective mitophagy in LPS-induced inflammation in macrophages and acute lung injury in mice. Together, our research indicates that MAFF is a potential target of ZCY020 and other lathyrane diterpenoids, and that PROTAC technology can be an innovative approach and a useful supplement for target identification of natural products.