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Microbial communities capable of hydrocarbon degradation linked to biosurfactant (BS) and bioemulsifier (BE) production are basically unexplored in the Gulf of México (GOM). In this work, the BS and BE production of culturable marine bacterial hydrocarbonoclasts consortia isolated from two sites (the Perdido Fold Belt and Coatzacoalcos area) was investigated. The prospection at different locations and depths led to the screening and isolation of a wide variety of bacterial consortia with BS and BE activities, after culture enrichment with crude oil and glycerol as the carbon sources. At least 55 isolated consortia presented reduction in surface tension (ST) and emulsifying activity (EI24 ). After colony purification, bacteria were submitted to polyphasic analysis assays that resulted in the identification of different strains of cultivable Gammaproteobacteria Gram (-) Citrobacter, Enterobacter, Erwinia, Pseudomonas, Vibrio, Shewanella, Thalassospira, Idiomarina, Pseudoalteromonas, Photobacterium, and Gram (+) Staphylococcus, Bacillus, and Microbacterium. Overall, the best results for ST reduction and EI24 were obtained with consortia. Individually, Pseudomonas, Bacillus, and Enterobacter strains showed the best results for the reduction of ST after 6 days, while Thalassospira and Idiomarina strains showed the best results for EI24 (above 68% after 9 days). Consortia isolates from the GOM had the ability to degrade crude oil by up to 40-80% after 24 and 36 months, respectively. In all cases, biodegradation of crude oil was related to the reduction in ST and bioemulsifying activity and was independent from the depth in the water column.

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Yeast surface display (YSD) is a "whole-cell" platform used for the heterologous expression of proteins immobilized on the yeast's cell surface. YSD combines the advantages eukaryotic systems offer such as post-translational modifications, correct folding and glycosylation of proteins, with ease of cell culturing and genetic manipulation, and allows of protein immobilization and recovery. Additionally, proteins displayed on the surface of yeast cells may show enhanced stability against changes in temperature, pH, organic solvents, and proteases. This platform has been used to study protein-protein interactions, antibody design and protein engineering. Other applications for YSD include library screening, whole-proteome studies, bioremediation, vaccine and antibiotics development, production of biosensors, ethanol production and biocatalysis. YSD is a promising technology that is not yet optimized for biotechnological applications. This mini review is focused on recent strategies to improve the efficiency and selection of displayed proteins. YSD is presented as a cutting-edge technology for the vectorial expression of proteins and peptides. Finally, recent biotechnological applications are summarized. The different approaches described herein could allow for a better strategy cascade for increasing protein/peptide interaction and production.

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The search for novel biosurfactants (Bs) requires the isolation of microorganisms from different environments. The Gulf of Mexico (GoM) is a geographical area active in the exploration and exploitation of hydrocarbons. Recent metagenomic and microbiologic studies in this area suggested a potential richness for novel Bs microbial producers. In this work, nineteen bacterial consortia from the GoM were isolated at different depths of the water column and marine sediments. Bs production from four bacterial consortia was detected by the CTAB test and their capacity to reduce surface tension (ST), emulsion index (EI24), and hemolytic activity. These bacterial consortia produced Bs in media supplemented with kerosene, diesel, or sucrose. Cultivable bacteria from these consortia were isolated and identified by bacterial polyphasic characterization. In some consortia, Enterobacter cloacae was the predominant specie. E. cloacae BAGM01 presented Bs activity in minimal medium and was selected to improve its Bs production using a Taguchi and Box-Behnken experimental design; this strain was able to grow and presented Bs activity at 35 g L-1 of NaCl. This Bs decreased ST to around 34.5 ± 0.56 mNm-1 and presented an EI24 of 71 ± 1.27%. Other properties of this Bs were thermal stability, stability in alkaline conditions, and stability at high salinity, conferring important and desirable characteristics in multiple industries. The analysis of the genome of E. cloacae BAGM01 showed the presence of rhlAB genes that have been reported in the synthesis of rhamnolipids, and alkAB genes that are related to the degradation of alkanes. The bioactive molecule was identified as a rhamnolipid after HPLC derivatization, 1H NMR, and UPLC-QTOF-MS analysis.

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Highly prized huperzine A (Hup A), a natural alkaloid formerly isolated from the Chinese medicinal plant Huperzia serrata, has been widely used for the treatment of Alzheimer disease, inspiring us to search for endophytic fungi that produce this compound. In this study, we obtained the C17 fungus isolate from the Mexican club moss Phlegmariurus taxifolius, which produced a yield of 3.2 µg/g Hup A in mycelial dry weight, when cultured in potato dextrose broth medium. The C17 isolate was identified as belonging to the genus Fusarium with reference to the colony´s morphological characteristics and the presence of macroconidia and microconidia structures; and this was confirmed by DNA-barcoding analysis, by amplifying and sequencing the ribosomal internal transcribed spacer (rITS).

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A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug-1 and 6.22 ± 0.53 gL-1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (µmol min-1) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol.

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Two laccase isoforms (lcc1 and lcc2) produced by Trametes versicolor, grown on oak sawdust under solid-state fermentation conditions, were purified and characterized. The two isoforms showed significant biochemical differences. Lcc1 and lcc2 had MWs of 60 and 100 kDa, respectively. Both isoforms had maximal activity at pH 3 with ABTS and 2,6-dimethyloxyphenol (DMP). Lcc1 was the most attractive isoform due to its greater affinity towards all the laccase substrates used. Lcc1 had Km values of 12, 10, 15 and 17 mM towards ABTS, DMP, guaiacol and syringaldazine, respectively. Lcc2 had equivalent values of 45, 47, 15 and 39 mM. The biochemical properties of lcc1 substantiate the potential of this enzyme for application in the treatment of contaminated water with low pH values and high phenolic content.

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Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.

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Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. loti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

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