Tuning Chemoenzymatic Pd/Laccase Conformation Toward Optimized Heterogeneous Aerobic Oxidation.

Heterogeneous chemoenzymatic catalysts differing in their spatial organization and relative orientation of their enzymatic laccase and Pd units confined into macrocellular silica foams were tested on veratryl alcohol oxidation. When operating under continuous flow, we show that the catalytic efficiency of hybrids is significantly enhanced when the Pd(II) complex is combined with a laccase exhibiting a surface located lysine next to the T1 oxidation site of the enzyme.

In vitro Applications of the Terpene Mini-Path 2.0.

In 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.

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.

Halomonas olivaria sp. nov., a moderately halophilic bacterium isolated from olive-processing effluents.

A moderately halophilic, Gram-stain-negative, non-sporulating bacterium designed as strain TYRC17(T) was isolated from olive-processing effluents. The organism was a straight rod, motile by means of peritrichous flagella and able to respire both oxygen and nitrate. Growth occurred with 0-25 % (w/v) NaCl (optimum, 7 %), at pH 5-11 (optimum, pH 7.0) and at 4-50 °C (optimally at 35 °C). It accumulated poly-ß-hydroxyalkanoate granules and produced exopolysaccharides. The predominant fatty acids were C18 : 1ω7c, C16 : 1ω7c and C16 : 0. Ubiquinone 9 (Q-9) was the only respiratory quinone. The DNA G+C content of TYRC17(T) was 53.9 mol%. Phylogenetic analyses of 16S rRNA gene sequences revealed that the strain represents a member of the genus Halomonas and more precisely of the subgroup containing Halomonas sulfidaeris, H. titanicae, H. variabilis, H. zhanjiangensis, H. alkaliantarctica, H. boliviensis and H. neptunia. TYRC17(T) showed high 16S-rRNA sequence identities in particular with the three last species listed (99.4-99.5 %). A multilocus sequence analysis (MLSA) using the 23S rRNA, gyrB, rpoD and secA genes allowed clarifying the phylogenetic position of TYRC17(T). This, combined with the level of DNA-DNA hybridization between TYRC17(T) and its closest relatives ranging from 21.6 % to 48.4 %, indicated that TYRC17(T) did not represent any of these species. On the basis of phenotypic and genotypic characteristics, and also genomic and phylogenetic evidence, it was concluded that strain TYRC17(T) represented a novel species of the genus Halomonas. The name Halomonas olivaria sp. nov. is proposed with TYRC17(T) ( = DSM 19074(T) = CCUG 53850B(T)) as the type strain.

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

This 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⊂UNIK157 ) or opposite position (1⊂UNIK71 ) 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⊂UNIK157 being twice as active than 1⊂UNIK71 (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.

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms.

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.

Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria.

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMO Xeno ) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMO Acineto ) in Synechocystis sp. PCC 6803 (25 vs 2.3 U gDCW -1 at an optical density of OD750 = 10) and an initial rate of 3.7 ± 0.2 mM h-1. While the cells containing CHMO Acineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMO Xeno , which was attributed to the much faster lactone formation and a 10-fold lower K M value of BVMO Xeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h-1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygen-dependent enzymatic processes.

Phylogenetic and genetic variation among Fe(II)-oxidizing acidithiobacilli supports the view that these comprise multiple species with different ferrous iron oxidation pathways.

Autotrophic acidophilic iron- and sulfur-oxidizing bacteria of the genus Acidithiobacillus constitute a heterogeneous taxon encompassing a high degree of diversity at the phylogenetic and genetic levels, though currently only two species are recognized (Acidithiobacillus ferrooxidans and Acidithiobacillus ferrivorans). One of the major functional disparities concerns the biochemical mechanisms of iron and sulfur oxidation, with discrepancies reported in the literature concerning the genes and proteins involved in these processes. These include two types of high-potential iron-sulfur proteins (HiPIPs): (i) Iro, which has been described as the iron oxidase; and (ii) Hip, which has been proposed to be involved in the electron transfer between sulfur compounds and oxygen. In addition, two rusticyanins have been described: (i) rusticyanin A, encoded by the rusA gene and belonging to the well-characterized rus operon, which plays a central role in the iron respiratory chain; and (ii) rusticyanin B, a protein to which no function has yet been ascribed. Data from a multilocus sequence analysis of 21 strains of Fe(II)-oxidizing acidithiobacilli obtained from public and private collections using five phylogenetic markers showed that these strains could be divided into four monophyletic groups. These divisions correlated not only with levels of genomic DNA hybridization and phenotypic differences among the strains, but also with the types of rusticyanin and HiPIPs that they harbour. Taken together, the data indicate that Fe(II)-oxidizing acidithiobacilli comprise at least four distinct taxa, all of which are able to oxidize both ferrous iron and sulfur, and suggest that different iron oxidation pathways have evolved in these closely related bacteria.

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

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-Mn2+ chemical autoxidation (PyoCHEM yield 0.317 g/g substrate), (2) an induced bacterial culture of Halomonas titanicae through the 4-hydroxyphenylacetic acid-1-hydroxylase route (PyoBACT, 0.55 g/L), and (3) a process using a recombinant laccase extract with the highest level produced (PyoENZ, 1.25 g/g substrate) and all the criteria for a large-scale prototype. The chemical structures had been investigated by 13C solid-state NMR (CP-MAS) and FTIR. Car-Car bindings predominated in the three polymers, Car-O-Car (ether) linkages being absent, proposing mainly C3-C6 (α-bindings) and C4-C6 (ß-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, PyoENZ exhibited an Mw of 5,400 Da, was hyperthermostable, non-cytotoxic even after irradiation, scavenged ROS induced by keratinocytes, and had a highly DPPH-antioxidant and Fe3+-reducing activity. As a representative pigment of living cells and an available standard, PyoENZ might also be useful for applications in extreme conditions and skin protection.

Integrated Strategy for Lead Optimization Based on Fragment Growing: The Diversity-Oriented-Target-Focused-Synthesis Approach.

Over the past few decades, hit identification has been greatly facilitated by advances in high-throughput and fragment-based screenings. One major hurdle remaining in drug discovery is process automation of hit-to-lead (H2L) optimization. Here, we report a time- and cost-efficient integrated strategy for H2L optimization as well as a partially automated design of potent chemical probes consisting of a focused-chemical-library design and virtual screening coupled with robotic diversity-oriented de novo synthesis and automated in vitro evaluation. The virtual library is generated by combining an activated fragment, corresponding to the substructure binding to the target, with a collection of functionalized building blocks using in silico encoded chemical reactions carefully chosen from a list of one-step organic transformations relevant in medicinal chemistry. The proof of concept was demonstrated using the optimization of bromodomain inhibitors as a test case, leading to the validation of several compounds with improved affinity by several orders of magnitude.

Bioconversion of tyrosol into hydroxytyrosol and 3,4-dihydroxyphenylacetic acid under hypersaline conditions by the new Halomonas sp. strain HTB24.

This paper reports the characterization of a Halomonas sp. strain (named HTB24) isolated from olive-mill wastewater and capable of transforming tyrosol into hydroxytyrosol (HT) and 3,4-dihydroxyphenylacetic acid (DHPA) in hypersaline conditions. This is the first time that a halophile has been shown to perform such reactions. The potent natural antioxidant HT was obtained through a C3 hydroxylation on the ring cycle, whereas DHPA was synthesized via the 4-hydroxyphenylacetic acid (HPA) pathway, which has been well described from other bacterial sources. HT was produced first, and then DHPA was detected in the medium accompanied by traces of HPA. HPA involved another pathway resulting from the activity of an aryl-dehydrogenase, which is suggested to be responsible for both tyrosol and hydroxytyrosol oxidation. Maximal HT content (2.30 mM) and maximal DHPA (5.15+/-0.42 mM) were obtained from a culture inoculated in the presence of 20 mM tyrosol and 0.5 g L(-1) yeast extract. Following this, DHPA was quickly degraded into 5-carboxymethyl-2-hydroxymuconic semialdehyde by a 2,3-dioxygenase, finally resulting in succinate and pyruvate. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain HTB24, with a G+C content of 55.3 mol%, is closely related to Halomonas neptunia DSM 15720(T), 'Halomonas alkaliantarctica' DSM 15686(T) and Halomonas boliviensis DSM 15516(T).

The Global Redox Responding RegB/RegA Signal Transduction System Regulates the Genes Involved in Ferrous Iron and Inorganic Sulfur Compound Oxidation of the Acidophilic Acidithiobacillus ferrooxidans.

The chemical attack of ore by ferric iron and/or sulfuric acid releases valuable metals. The products of these reactions are recycled by iron and sulfur oxidizing microorganisms. These acidophilic chemolithotrophic prokaryotes, among which Acidithiobacillus ferrooxidans, grow at the expense of the energy released from the oxidation of ferrous iron and/or inorganic sulfur compounds (ISCs). In At. ferrooxidans, it has been shown that the expression of the genes encoding the proteins involved in these respiratory pathways is dependent on the electron donor and that the genes involved in iron oxidation are expressed before those responsible for ISCs oxidation when both iron and sulfur are present. Since the redox potential increases during iron oxidation but remains stable during sulfur oxidation, we have put forward the hypothesis that the global redox responding two components system RegB/RegA is involved in this regulation. To understand the mechanism of this system and its role in the regulation of the aerobic respiratory pathways in At. ferrooxidans, the binding of different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms of RegA) on the regulatory region of different genes/operons involved in ferrous iron and ISC oxidation has been analyzed. We have shown that the four RegA forms are able to bind specifically the upstream region of these genes. Interestingly, the phosphorylation of RegA did not change its affinity for its cognate DNA. The transcriptional start site of these genes/operons has been determined. In most cases, the RegA binding site(s) was (were) located upstream from the -35 (or -24) box suggesting that RegA does not interfere with the RNA polymerase binding. Based on the results presented in this report, the role of the RegB/RegA system in the regulation of the ferrous iron and ISC oxidation pathways in At. ferrooxidans is discussed.

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.

Hydroxytyrosol (HTyr) is a potent natural antioxidant found in olive mill wastewaters. Bacterial conversion of 4-tyrosol (2-(4-hydroxyphenyl)-ethanol) to HTyr was reported in a limited number of bacterial species including Pseudomonas aeruginosa. In this work, we studied this conversion, taking as a model the newly isolated Halomonas sp. strain HTB24. It was first hypothesized that the enzyme responsible for 4-tyrosol hydroxylation in HTyr was a 4-hydroxyphenylacetic acid 3-hydroxylase (HPAH, EC 1.14.13.3), previously known to convert 4-hydroxyphenylacetic acid (4-HPA) into 3,4-dihydroxyphenylacetic acid (3,4-DHPA) in P. aeruginosa. Cloning and expression of hpaB (oxygenase component) and hpaC (reductase component) genes from P. aeruginosa confirmed this hypothesis. Furthermore, using cultures of HTB24 containing 4-tyrosol, it was shown that 4-HPA accumulation preceded 4-tyrosol hydroxylation. We further demonstrated that the synthesis of HPAH activity was induced by 4-HPA, with the latter compound being formed from 4-tyrosol oxidation by aryl-dehydrogenases. Interestingly, similar results were obtained with other 4-HPA-induced bacteria, including P. aeruginosa, Serratia marcescens, Escherichia coli, Micrococcus luteus and other Halomonas, thus demonstrating general hydroxylating activity of 4-tyrosol by the HPAH enzyme. E. coli W did not have aryl-dehydrogenase activity and hence were unable to oxidize 4-tyrosol to 4-HPA and HTyr to 3,4-DHPA, making this bacterium a good candidate for achieving better HTyr production.