RESUMO
Water-filled sinkholes known locally as cenotes, found on the Yucatán Peninsula, have remarkable biodiversity. The primary objective of this study was to explore the biotechnological potential of Gram-positive cultivable bacteria obtained from sediment samples collected at the coastal cenote Pol-Ac in Yucatán, Mexico. Specifically, the investigation aimed to assess production of hydrolytic enzymes and antimicrobial compounds. 16 S rRNA gene sequencing led to the identification of 49 Gram-positive bacterial isolates belonging to the phyla Bacillota (n = 29) and Actinomycetota (n = 20) divided into the common genera Bacillus and Streptomyces, as well as the genera Virgibacillus, Halobacillus, Metabacillus, Solibacillus, Neobacillus, Rossellomorea, Nocardiopsis and Corynebacterium. With growth at 55ºC, 21 of the 49 strains were classified as moderately thermotolerant. All strains were classified as halotolerant and 24 were dependent on marine water for growth. Screening for six extracellular hydrolytic enzymes revealed gelatinase, amylase, lipase, cellulase, protease and chitinase activities in 93.9%, 67.3%, 63.3%, 59.2%, 59.2% and 38.8%, of isolated strains, respectively. The genes for polyketide synthases type I, were detected in 24 of the strains. Of 18 strains that achieved > 25% inhibition of growth in the bacterial pathogen Staphylococcus aureus ATCC 6538, 4 also inhibited growth in Escherichia coli ATCC 35,218. Isolates Streptomyces sp. NCA_378 and Bacillus sp. NCA_374 demonstrated 50-75% growth inhibition against at least one of the two pathogens tested, along with significant enzymatic activity across all six extracellular enzymes. This is the first comprehensive report on the biotechnological potential of Gram-positive bacteria isolated from sediments in the cenotes of the Yucatán Peninsula.
Assuntos
Biodiversidade , Sedimentos Geológicos , Bactérias Gram-Positivas , RNA Ribossômico 16S , Sedimentos Geológicos/microbiologia , México , Bactérias Gram-Positivas/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/classificação , RNA Ribossômico 16S/genética , Bioprospecção , Filogenia , Antibacterianos/farmacologia , Água do Mar/microbiologiaRESUMO
Introduction: Biofouling poses a significant economic threat to various marine industries, leading to financial losses that can reach billions of euros annually. This study highlights the urgent need for effective alternatives to traditional antifouling agents, particularly following the global ban on organotin compounds. Material and methods: Streptomyces aculeolatus PTM-346 was isolated from sediment samples on the shores of the Madeira Archipelago, Portugal. The crude extract was fractionated using silica flash chromatography and preparative HPLC, resulting in two isolated marinone compounds: madeirone (1), a novel marinone derivative discovered in this study, and neomarinone (2). The antifouling activities of these compounds were tested against five marine bacterial species and the larvae of the mussel Mytilus galloprovincialis. Additionally, in silico and in vivo environmental toxicity evaluations of madeirone (1) and neomarinone (2) were conducted. Results: Madeirone (1) demonstrated significant antibiofilm efficacy, inhibiting Phaeobacter inhibens by up to 66%, Marinobacter hydrocarbonoclasticus by up to 60%, and Cobetia marina by up to 40%. Neomarinone (2) also exhibited substantial antibiofilm activity, with inhibition rates of up to 41% against P. inhibens, 40% against Pseudo-oceanicola batsensis, 56% against M. hydrocarbonoclasticus, 46% against C. marina, and 40% against Micrococcus luteus. The growth inhibition activity at the same concentrations of these compounds remained below 20% for the respective bacteria, highlighting their effectiveness as potent antibiofilm agents without significantly affecting bacterial viability. Additionally, both compounds showed potent effects against the settlement of Mytilus galloprovincialis larvae, with EC50 values of 1.76 µg/mL and 0.12 µg/mL for compounds (1) and (2), respectively, without impairing the viability of the targeted macrofouling species. In silico toxicity predictions and in vivo toxicity assays both support their potential for further development as antifouling agents. Conclusion: The newly discovered metabolite madeirone (1) and neomarinone (2) effectively inhibit both micro- and macrofouling. This distinct capability sets them apart from existing commercial antifouling agents and positions them as promising candidates for biofouling prevention. Consequently, these compounds represent a viable and environmentally friendly alternative for incorporation into paints, primers, varnishes, and sealants, offering significant advantages over traditional copper-based compounds.
RESUMO
Glycerol dehydrogenase (GldA) from Escherichia coli BW25113, naturally catalyzes the oxidation of glycerol to dihydroxyacetone. It is known that GldA exhibits promiscuity towards short-chain C2-C4 alcohols. However, there are no reports regarding the substrate scope of GldA towards larger substrates. Herein we demonstrate that GldA can accept bulkier C6-C8 alcohols than previously anticipated. Overexpression of the gldA gene in the knockout background, E. coli BW25113 ΔgldA, was strikingly effective converting 2 mM of the compounds: cis-dihydrocatetechol, cis-(1 S,2 R)- 3-methylcyclohexa-3,5-diene-1,2-diol and cis-(1 S,2 R)- 3-ethylcyclohexa-3,5-diene-1,2-diol, into 2.04 ± 0.21 mM of catechol, 0.62 ± 0.11 mM 3-methylcatechol, and 0.16 ± 0.02 mM 3-ethylcatechol, respectively. In-silico studies on the active site of GldA enlightened the decrease in product formation as the steric substrate demand increased. These results are of high interests for E. coli-based cell factories expressing Rieske non-heme iron dioxygenases, producing cis-dihydrocatechols, since such sough-after valuable products can be immediately degraded by GldA, substantially hampering the expected performance of the recombinant platform.
Assuntos
Dioxigenases , Desidrogenase do Álcool de Açúcar , Escherichia coli/genética , Escherichia coli/metabolismo , Desidrogenase do Álcool de Açúcar/genética , Dioxigenases/metabolismo , Oxirredução , Glicerol/metabolismoRESUMO
Phosphorous-NMR is scarcely employed to evaluate enzyme kinetics of kinase driven monophosphorylations, despite of being a powerful and reliable tool to undoubtedly detect the actual phosphoryl transfer to the targeted substrate. Another advantage is that an external supplementation source of the NMR active isotope is not required, since 31P is highly abundant in nature. Glycerol kinase (GlpK) from E. coli is an exemplary ATP-dependent kinase/phosphotransferase model to illustrate the value and usefulness of a 31P-NMR-based approach to assess the enzymatically driven monophosphorylation of glycerol. Moreover, the described approach offers an alternative to the indirect coupled glycerol kinase enzyme assays. Herein, we provided a real time 31P-NMR-based method customized for the direct assessment of the glycerol kinase enzyme activity.â¢Real-time detection for phosphoryl group dynamics in the GlpK driven reactionâ¢Direct assessment of product formation (glycerol-monophosphate)â¢Parallel determination of cosubstrate (ATP) consumption and coproduct (ADP) generationâ¢Method validation was performed via 31P-NMR for each phosphorylated molecule involved in the reaction in order to assist in the molecular assignments.
RESUMO
Rieske non-heme iron dioxygenases are a class of intriguing enzymes covering a broad reaction and substrate spectrum and have been studied extensively in the last decades. In nature, these biocatalysts are essential for the production of cis-dihydroxylated metabolites, as a first step during the degradation of aromatic compounds in microorganisms. The enzymes are able to produce relevant amounts of compounds in short reaction times, but the effort for constant cultivation of recombinant cells and production of cell mass for biotransformations is high. To overcome the steady production process, our task was to find a way to make the biocatalysts durable and storable. In this way, laboratories lacking equipment for microbiology, e.g. chemistry laboratories, can be supplied with the enzymes to open up new possibilities in the production of molecules. We present a quick and efficient method that uses lyophilization to freeze-dry recombinant whole-cells that harbor the enzyme of interest. By washing the cells with a cryoprotectant before lyophilization, we could conserve the enzyme activity to the level of freshly harvested cells. Moreover, this simple to apply method enables subsequent steps like storage of the cell powder for transportation and on demand use in biotransformations. The method was established with the cumene dioxygenase (CDO) of Pseudomonas fluorescens IP01 and its variant CDO M232A expressed in E. coli JM109 (DE3) cells, employing R-limonene and naphthalene, respectively, as substrates in biotransformations. The method could be successfully applied in the analytical and semi-preparative reaction scale.â¢Preservation of biocatalysts in recombinant whole-cells.â¢Ready-to-use enzymatic reaction.â¢Semi-preparative biotransformation with lyophilized whole-cells.
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In an attempt to establish a biosynthetic route towards isobutene, we faced the problem that the first intermediate isobutyl-monophosphate was not commercially available. In order to overcome this limitation we searched in the literature for protocols reporting the synthesis of phosphate monoesters from alcohols. Based on the suitability of the preceding developments for our purposes, we established a customized method for the fast, easy and affordable generation of the pursued molecule. Herein, a prompt and straightforward method for isobutyl-monophosphate (ammonium salt) is provided.â¢This is a customized method for the production of isobutyl-monophosphate (ammonium salt), using isobutanol as starting compoundâ¢Synthesis takes place in a one-pot fashion, under mild reaction conditions, in 2 hâ¢The established sequential strategy requires 8 h at the most, including synthesis and purification steps to obtain the isolated product.
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cis-1,2-Dihydro-1,2-naphthalenediol (DHND) is a valuable molecule employed for the pharmaceutical synthesis of bioactive compounds, such as bicyclic conduritol analogues. Enantiopure (+)-(1R,2S)-DHND (>98 % ee) is easily biosynthesized through the dearomatizing dihydroxylation of naphthalene, catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1. However, the opposite enantiomer (-)-(1S,2R)-DHND could not be directly accessed, neither by chemical synthesis nor via biocatalytic approaches. Herein, we report a one-step biosynthesis of the opposite enantiomer (-)-(1S,2R)-DHND in a recombinant TDO E. coli BW25113 platform. We based on a semi-rational approach to generate a set of TDO variants, targeting exclusively the hotspot position F366, in order to enable an enantiomeric switch in the generated product. Eight out of nine single point variants were active and showed not only an alteration in enantioselectivity, but also generated an enantiomeric excess of the pursued product. Variant TDOF366V outperformed above the rest of the set, enabling the synthesis of (-)-(1S,2R)-DHND not only with an excellent enantiomeric excess of 90 %, but also with an advantageous product formation. A comparative semi-preparative biosynthesis yielded, 287 mg of (+)-(1R,2S)-DHND (>98 % ee) and 101 mg of (-)-(1S,2R)-DHND (90 % ee), when performed in a total volume of 100 mL with TDO wild-type and TDOF366V resting cells, respectively.
Assuntos
Escherichia coli , Pseudomonas putida , Escherichia coli/genética , Naftóis , Oxigenases , Pseudomonas putida/genéticaRESUMO
The compound cis-1,2-dihydrocatechol (DHC) is highly valuable since it finds wide application in the production of fine chemicals and bioactive compounds with medical relevance. The biotechnological process to generate DHC involves a dearomatizing dihydroxylation reaction catalyzed by toluene dioxygenase (TDO) from P. putida F1, employing benzene as substrate. We aimed to enhance the biotechnological E. coli BW25113 platform for DHC production by identifying the key operational parameters positively influencing the final isolated yield. Thereby, we observed an unreported downstream reaction, generating catechol from DHC, affecting, in a negative manner, the final titer for the product. Expression temperature for the TDO-system showed to have the highest influence in terms of final isolated yield. A KEIO-collection-based screening approach highlighted glycerol dehydrogenase (GldA) as the main responsible enzyme for the undesired reaction. We transferred the TDO-system to E. coli BW25113 ΔgldA and applied the enhanced operational set-up on it. This enhanced platform enabled the production of 1.41â¯g L-1 DHC in isolated yield, which represents a two-fold increase compared with the starting working conditions. To our knowledge, this is the highest DHC production accomplished in recombinant E. coli at semi-preparative scale, providing a robust and accessible biotechnological platform for DHC synthesis.
Assuntos
Pseudomonas putida , Catecóis , Escherichia coli/genética , Oxigenases , Desidrogenase do Álcool de AçúcarRESUMO
cis-Dihydrodiendiols are valuable compounds, finding multiple application as chiral synthons in organic chemistry. The biotechnological route for the generation of cis-dihydrodiendiols involves the dihydroxylation of aromatic compounds, catalyzed by Rieske non-heme iron dioxygenases. To date, numerous examples of recombinant E. coli, harboring such dioxygenases, can be found in the literature. Nevertheless, there is only a minor number of publications, addressing the E. coli catalyzed degradation of cis-dihydrodiendiols into catechols via dehydrogenases. Identification and elimination of such dehydrogenase catalyzed degradation is key for the establishment of enhanced recombinant E. coli platforms pursuing the production of cis-dihydrodiendiols. Here, we provide a fast and easy strategy for the identification of promiscuous alcohol dehydrogenases in E. coli BW25113, catalyzing the degradation of cis-dihydrodiendiols into catechols. This approach is based on the screening of dehydrogenase deficient KEIO strains, regarding their incapability of degrading a cis-dihydrodiendiol of choice.â¢Novel screening strategy for E. coli BW25113 dehydrogenase knock-outs, incapable of degrading cis-dihydrodiendiols was validated for cis-1,2-dihydrocatechol as substrateâ¢Corresponding knock-outs can be used for recombinant production of cis-dihydrodiendiolsâ¢Simple analysis based on liquid chromatography with diode array detector (HPLC-DAD).
RESUMO
Rieske non-heme iron dioxygenases (ROs) are promising candidates to perform dihydroxylation reactions, since they are capable to incorporate both atoms of molecular oxygen into vicinal non-activated CH bonds, endowing valuable products for pharmaceutical and chemical applications. ROs harbor attractive features such as, striking activity in combination with remarkable regio- and stereo-selectivity, wide reaction spectrum, and broad substrate scope. In order to identify, characterize, and enhance targeted features of dioxygenases and related oxygen dependent enzymes via enzyme engineering and evolution approaches, proper screening and analytical methods are essential to detect and to analyze the expected dihydroxylation activity. This chapter presents different methodologies suitable for the study of dihydroxylation reactions. Detailed descriptions of our established analytical protocols for both gas and liquid chromatography, as well as a colorimetric assay to detect dioxygenase activity are provided. In addition, a novel and reliable system for real-time detection of oxygen consumption, in vivo, is reported.