Filamentous fungi, although producing noxious molecules such as mycotoxins, have been used to produce numerous drugs active against human diseases such as paclitaxel, statins, and penicillin, saving millions of human lives. Cyclodepsipeptides are fungal molecules with potentially adverse and positive effects. Although these peptides are not novel, comparative studies of their antimicrobial activity, toxicity, and mechanism of action are still to be identified. In this study, the fungal cyclohexadepsipeptides enniatin (ENN) and beauvericin (BEA) were assessed to determine their antimicrobial activity and cytotoxicity against human cells. Results showed that these peptides were active against Gram-positive bacteria, Mycobacterium, and fungi, but not against Gram-negative bacteria. ENN and BEA had a limited hemolytic effect, yet were found to be toxic at low doses to nucleated human cells. Both peptides also interacted with bacterial lipids, causing low to no membrane permeabilization, but induced membrane depolarization and inhibition of macromolecules synthesis. The structure–activity analysis showed that the chemical nature of the side chains present on ENN and BEA (either iso-propyl, sec-butyl, or phenylmethyl) impacts their interaction with lipids, antimicrobial action, and toxicity.
All naturally produced terpenes are derived from two universal C5 diphosphate precursors, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Various prenyl transferases use DMAPP to prenylate aromatic compounds, while others, in combination with IPP, lead to the enzymatic formation of geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranylfarnesyl diphosphate, the direct precursors of monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and sesterterpenes (C25), respectively. FPP and GGPP are also the basis for the biosynthesis of triterpenes (steroids) and tetraterpenes (carotenoids), respectively. Nature has developed two biosynthetic pathways to produce DMAPP and IPP, the mevalonate (MEV) pathway and the methylerythritol phosphate (MEP) pathway. Both use compounds derived from glucose through glycolysis, and 18 enzymes are involved to generate both DMAPP and IPP. Here, we sought to simplify biochemical access to these two universal diphosphates using the two commercially and industrially available C5-OHs, dimethylallyl alcohol and isopentenol (IOH), as starting substrates, as well as two enzymes, selected from a diverse choice, able to carry out the double phosphorylation of these two C5-OHs at room temperature using ATP as a phosphate donor. The first phosphorylation is performed by a promiscuous acid phosphatase (AP), used in the reverse reaction mode, whereas the second is performed by the recently described isopentenyl phosphate kinase (IPK). We show the interest of this artificial biosynthetic terpene mini-path (TMP) by testing it in a three-enzyme cascade, leading to the formation of the cytotoxic prenylated diketopiperazine tryprostatin B (TB) from chemically synthesized brevianamide F (BF), using FtmPT1 prenyltransferase as a biocatalyst, in addition to the two previously mentioned kinases. We first performed the proof of concept of this simplified pathway in vivo (Escherichia coli), using already described enzymes, that is, an AP from Salmonella enterica and an IPK from Thermoplasma acidophilum. The complete conversion of BF (3.3 mM, 1 g/L) to TB was obtained after optimization of culture conditions and process parameters. Following this first success, we performed a screen in search of highly active phosphatases and IPKs to develop the TMP in vitro. A highly active AP from Xanthomonas translucens and an IPK from Methanococcus vannielii were selected from these screens, allowing the in vitro development of the TMP. Under optimized conditions, the three-enzyme cascade led to the total transformation of BF (10 mM, 3.3 g/L) to TB in less than 24 h, establishing the in vitro utility as well as the in vivo utility of the TMP. The implementation of this biosynthetic TMP offers thus the possibility to access virtually any terpene structure using two easily commercially and industrially available compounds in bulk, either in vivo or in vitro, and is thus a viable alternative to the natural MEV and MEP pathways for bioaccess to terpenes.
Tyrosinases act in the development of organoleptic properties of tea, raisins, etc., but also cause unwanted browning of fruits, vegetables, and mushrooms. The tyrosinase from Agaricus bisporus has been used as a model to study tyrosinase inhibitors, which are also indispensable in the treatment of skin pigmentation disorders. However, this model has disadvantages such as side enzyme activities and the presence of multiple isoenzymes. Therefore, we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme. 2'- and 3'-hydroxyaurones acted as its activators and 2',4'-dihydroxyaurone as an inhibitor, whereas 4'-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase effector studies, being a single isoenzyme and void of side enzyme activities.
Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.
The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.
The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε‐caprolactone and 3,4‐dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi‐enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter‐balanced by a higher concentration. Using a preconception‐free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1‐indanol (7.5 mM) at a 0.5‐L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε‐caprolactone, for the productivity of 244 mg·L −1·h −1) and that for 1‐indanol 60% (0.3 g 3,4‐dihydrocoumarin, for the productivity of 140 mg·L −1·h −1).
We report the unprecedented use of laccase to functionalize phenol-modified carbon nanotubes (CNTs). Enzymatically-generated metallopolyphenols or the laccase itself can be softly and efficiently immobilized using the ability of laccase...
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