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A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18‐/14‐membered sesterterpenoids including five new ones and two 14‐membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3‐ and 2.5‐fold, respectively, more sesterterpenoids than the wild‐type enzyme were engineered. Both 18‐ and 14‐membered ring sesterterpenoids displayed significant inhibitory activity on the IL‐2 and IFN‐γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well‐trodden territory of plant terpenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: Eupatorium adenophorum is a malignant invasive plant possessing extraordinary defense potency, but its chemical weaponry and formation mechanism have not yet been extensively investigated.We identified six cadinene sesquiterpenes, including two volatiles (amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol) and four nonvolatiles (9‐oxo‐10,11‐dehydroageraphorone, muurol‐4‐en‐3,8‐dione, 9‐oxo‐ageraphorone and 9β‐hydroxy‐ageraphorone), as the major constitutive and inducible chemicals of E. adenophorum. All cadinenes showed potent antifeedant activity against a generalist insect Spodoptera exigua, indicating that they have significant defensive roles.We cloned and functionally characterized a sesquiterpene synthase from E. adenophorum (EaTPS1), catalyzing the conversion of farnesyl diphosphate to amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol, which were purified from engineered Escherichia coli and identified by extensive nuclear magnetic resonance (NMR) spectroscopy. EaTPS1 was highly expressed in the aboveground organs, which was congruent with the dominant distribution of cadinenes, suggesting that EaTPS1 is likely involved in cadinene biosynthesis. Mechanical wounding and methyl jasmonate negatively regulated EaTPS1 expression but caused the release of amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol. Nicotiana benthamiana transiently expressing EaTPS1 also produced amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol, and showed enhanced defense function.The findings presented here uncover the role and formation of the chemical defense mechanism of E. adenophorum – which probably contributes to the invasive success of this plant – and provide a tool for manipulating the biosynthesis of biologically active cadinene natural products. [ABSTRACT FROM AUTHOR]

A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3- and 2.5-fold, respectively, more sesterterpenoids than the wild-type enzyme were engineered. Both 18- and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well-trodden territory of plant terpenoid biosynthesis., (© 2021 Wiley-VCH GmbH.)

(-)-5-Epieremophilene, an epimer of the versatile sesquiterpene (+)-valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1-3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (-)-5-epieremophilene production with yields of 42.4-76.0 mg/L in shake-flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1-FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (-)-5-epieremophilene were achieved. Biological assay indicated that (-)-5-epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC 50 =1.25 μg/cm 2 ), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (-)-5-epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology., (© 2020 Wiley-VHCA AG, Zurich, Switzerland.)

The genomic DNA of housefly larvae was extracted after ultrasound induction, and the structure was analyzed by UV, fluorescence, IR and 1H NMR. The 3'-end of attacin gene was sequenced and compared by means of PCR. All the results indicated that ultrasound induction can destroy the second structure and the base stacking of genomic DNA of housefly larvae, which will result in mismatch repair during DNA duplication and finally change the sequence of DNA, but it has no significant effect on chemical groups and chemical band of genomic DNA.

The antimicrobial peptides of insect are the main components of their non-specific immune system, and play a major role in the defense against the foreign disease-related microbes. In this report, a full length cDNA of Attacin, an insect antimicrobial peptide was cloned from housefly (Musca domestica) by homology cloning approach in combine with 3' and 5' RACE. Sequence analysis and phylogenetical study showed that this cDNA contained 778 nucleotides, with a 627 bp open reading frame (ORF) flanked with a 44 bp 5'UTR and a 107 bp 3'UTR. The encoded 208 amino acids housefly Attacin shared a high similarity of 50%-70% with that of the other dipterous insects. In addition, the phylogenetical analysis also indicated that the Attacin from housefly was in the same branch with those of other species, suggesting that they come from the same ancestor. The expression of Attacin transcript was measured by semi-quantitive RT-PCR. The results demonstrated that the expression of housefly Attacin is inducible, and that the level varies with the time of induction and the kinds of pathogens.