Study of a hexane-degrading consortium in a biofilter and in liquid culture : biodiversity, kinetics and characterization of degrading strains

A gasoline-degrading consortium, originating from a Mexican soil, was used to study its hexane-degradation kinetics in liquid culture and in a biofilter with mineral support. The biodiversity of the consortium depending on the culture conditions and electron and energy source (gasoline, hexane in liquid or hexane in the biofilter) was analyzed using a 16S rRNA-based approach. Significant differences between the populations were observed, indicating a probable adaptation to the substrate. Two strains, named SP2B and SP72-3, isolated from the consortium, belonged to Actinomycetes and demonstrated a high metabolic potential in hexane degradation. Even though the SP2B strain was related to Rhodococcus ruber DSM 43338(T) by phylogenetic studies, it displayed enlarged metabolic properties in hexane and other short-alkane degradation compared with the collection strain

Hydroxytyrosol from tyrosol using hydroxyphenylacetic acid-induced bacterial cultures and evidence of the role of 4-HPA 3-hydroxylase

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Identification of different alkane hydroxylase systems in Rhodococcus ruber strain SP2B, an hexane-degrading actinomycete

Aims: To investigate the alkane-hydroxylating system of isolate SP2B, closely related to Rhodococcus ruber DSM 43338T and uncharacterized so far for its alkane degradation genes. Methods and Results: Although isolate SP2B and reference strain can grow on by-products from hexane degradation, the type strain R. ruber was unable, unlike SP2B isolate, to use short-chain alkanes, as assessed by gas chromatography. Using PCR with specific or degenerated primers, inverse PCR and Southern blot, two alkane hydroxylase encoding genes (alkB) were detected in both bacteria, which is in agreement with their alkane range. The first AlkB was related to Rhodococcus AlkB7 enzymes and contains a nonbulky residue at a specific position, suggesting it might be involved in medium- and long-chain alkane oxidation. The second partial alkB gene potentially belongs to alkB5-type, which was found in bacteria unable to use hexane. Moreover, a partial P450 cytochrome alkane hydroxylase, thought to be responsible for the hexane degradation, was detected only in the isolated strain. Conclusions: Rhodococcus ruber SP2B should prove to be a promising candidate for bioremediation studies of contaminated sites because of its large degradation range of alkanes. Significance and Impact of the Study: This is the first thorough study on R. ruber alkane degradation systems

Production and properties of non-cytotoxic pyomelanin by laccase and comparison to bacterial and synthetic pigments

International audienceAbstract Pyomelanin is a polymer of homogentisic acid synthesized by microorganisms. This work aimed to develop a production process and evaluate the quality of the pigment. Three procedures have been elaborated and optimized, (1) an HGA-Mn 2+ chemical autoxidation (Pyo CHEM yield 0.317 g/g substrate), (2) an induced bacterial culture of Halomonas titanicae through the 4-hydroxyphenylacetic acid-1-hydroxylase route (Pyo BACT , 0.55 g/L), and (3) a process using a recombinant laccase extract with the highest level produced (Pyo ENZ , 1.25 g/g substrate) and all the criteria for a large-scale prototype. The chemical structures had been investigated by 13 C solid-state NMR (CP-MAS) and FTIR. C ar –C ar bindings predominated in the three polymers, C ar –O–C ar (ether) linkages being absent, proposing mainly C 3 -C 6 (α-bindings) and C 4 -C 6 (β-bindings) configurations. This work highlighted a biological decarboxylation by the laccase or bacterial oxidase(s), leading to the partly formation of gentisyl alcohol and gentisaldehyde that are integral parts of the polymer. By comparison, Pyo ENZ exhibited an M w of 5,400 Da, was hyperthermostable, non-cytotoxic even after irradiation, scavenged ROS induced by keratinocytes, and had a highly DPPH-antioxidant and Fe 3+ -reducing activity. As a representative pigment of living cells and an available standard, Pyo ENZ might also be useful for applications in extreme conditions and skin protection

Controlled Immobilization of a Palladium Complex/Laccase Hybrid into a Macrocellular Siliceous Host

International audienceThis study investigates the site-directed immobilization of a hybrid catalyst bearing a biquinoline-based-Pd(II) complex (1) and a robust laccase within cavities of a silica foam to favor veratryl alcohol oxidation. We performed the grafting of 1 at a unique surface located lysine of two laccase variants, either at closed (1⊂UNIK 157) or opposite position (1⊂UNIK 71) of the enzyme oxidation site. After immobilization into the cavities of silica monoliths bearing hierarchical porosity, we show that catalytic activity is dependent on the orientation and loading of each hybrid, 1⊂UNIK 157 being twice as active than 1⊂UNIK 71 (203 TON vs 100 TON) when operating under continuous flow. These systems can be reused 5 times, with an operational activity remaining as high as 40 %. We show that the synergy between 1 and laccase can be tuned within the foam. This work is a proof of concept for controlling the organization of a heterogeneous hybrid catalyst using a Pd/laccase/silica foam

In vitro Applications of the Terpene Mini‐Path 2.0

International audienceIn 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes

The Multifunctional Sactipeptide Ruminococcin C1 Displays Potent Antibacterial Activity In Vivo as Well as Other Beneficial Properties for Human Health

International audienceThe 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

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms

Summary: In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1–C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms