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Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

The molecular mechanisms underlying the development and progression of colorectal cancer (CRC) have not been clarified. The purpose of the present study was to identify key genes that may serve as novel therapeutic targets or prognostic predictors in patients with CRC using bioinformatics analysis. Four gene expression datasets were downloaded from the Gene Expression Omnibus database, which revealed 19 upregulated and 34 downregulated differentially expressed genes (DEGs). The downregulated DEGs were significantly enriched in eight pathways according to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. A protein-protein interaction network was constructed with 52 DEGs and 458 edges. Ten key genes were identified according to the degree value, betweenness centrality and closeness centrality. Survival analysis revealed that low expression of four of the ten genes, carcinoembryonic antigen related cell adhesion molecule 7 (CEACAM7), solute carrier family 4 member 4 (SLC4A4), glucagon (GCG) and chloride channel accessory 1 (CLCA1) genes, were associated with unfavorable prognosis in CRC. Furthermore, gene set enrichment analysis revealed that two pathways were significantly enriched in the CEACAM7 low-expression group. Thus, CEACAM7, SLC4A4, GCG and CLCA1 may be prognostic markers or therapeutic targets of CRC. Low CEACAM7 expression may be associated with the activation of glycosaminoglycan biosynthesis-chondroitin sulfate and extracellular matrix receptor interaction pathways and may affect the prognosis of CRC.

Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase with mono(per)oxygenase activity for the selective oxyfunctionalization of C-H bonds. Fueled by catalytic concentrations of H2O2, which acts as both oxygen donor and as final electron acceptor, this stable, soluble and extracellular enzyme is a potential biocatalyst for dozens of transformations that are of considerable interest in organic synthesis. In this chapter we describe the main attributes of this versatile enzyme, while reflecting on the directed evolution campaigns recently followed in our laboratory that set out to enhance the functional expression of UPO in yeast and improve the activity, as well as approximating its properties to the required industrial standards.

Versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems. With a broad substrate scope and minor requirements, VP is an extremely attractive blueprint to be designed by the directed evolution tool-box. In recent years, improved VP variants have been generated that meet industrial requirements in terms of improved heterologous functional expression and activity, as well as stability at high temperatures or in the presence of strong inhibitors. This review describes the making of VP by directed evolution, addressing the most important findings and challenges that were faced, along with the future prospects for this research field.

Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (~8. mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217. mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.

[EN] Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate >green catalysts>. This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts.

Interested by the relation of function and fashion expression, this project choose ‘carry’ as an example function to dive in. It explores how ‘carry’ affects the expression of wearing and how it can be interpret into fashion expression. The research question - what do we carry in life - gives the foundation in the developing of an aesthetic for sustaining. Problem solving was the methodology that I adopted from Industrial design. Together with other methods, it helped me in deign rationale. During the exploration, ‘carry’ is analyzed, decomposed and translated into a conceptual collection. The interpretation of ‘carry’ is not functional oriented or literal-symbol inspired. The dynamic moment of functioning is more of the focus rather than a static expression of garment. In addition, minimized fashion expression is also discussed as the aesthetic of sustainability., Program: Modedesignutbildningen

Interested by the relation of function and fashion expression, this project choose ‘carry’ as an example function to dive in. It explores how ‘carry’ affects the expression of wearing and how it can be interpret into fashion expression. The research question - what do we carry in life - gives the foundation in the developing of an aesthetic for sustaining. Problem solving was the methodology that I adopted from Industrial design. Together with other methods, it helped me in deign rationale. During the exploration, ‘carry’ is analyzed, decomposed and translated into a conceptual collection. The interpretation of ‘carry’ is not functional oriented or literal-symbol inspired. The dynamic moment of functioning is more of the focus rather than a static expression of garment. In addition, minimized fashion expression is also discussed as the aesthetic of sustainability., Program: Modedesignutbildningen

Itaconic acid is an important building block for the chemical industry. Currently, Aspergillus terreus is the main organism used for itaconic acid production. Due to the enormous citric acid production capacity of Aspergillus niger, this host is investigated as a potential itaconic acid production host. Several strategies have been tried so far: fermentation optimization, expression of cis-aconitate decarboxylase (cadA) alone and in combination with aconitase targeted to the same compartment, chassis optimization, and the heterologous expression of two transporters flanking the cadA gene. We showed that the heterologous expression of these two transporters were key to improving itaconic acid production in an A. niger strain that was unable to produce oxalic acid and gluconic acid. The expression of transporters has increased the production levels of other industrially relevant processes as well, such as ß-lactam antibiotics and bioethanol. Thus far, the role of transporters in production process optimization is a bit overlooked.

Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding., QC 20140613

[Background] Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory., [Results] A blood-active laccase (ChU-B mutant) fused to the native/evolved α-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the α-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers., [Conclusions] The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.

Differential localization and regulation of two aquaporin-1 homologs in the intestinal epithelia of the marine teleost Sparus aurata. Am J Physiol Regul Integr Comp Physiol 294: R993–R1003, 2008. First published January 2, 2008; doi:10.1152/ajpregu.00695.2007.—Aquaporin (AQP)-mediated intestinal water absorption may play a major osmoregulatory role in euryhaline teleosts, although the molecular identity and anatomical distribution of AQPs in the fish gastrointestinal tract is poorly known. Here, we have investigated the functional properties and cellular localization in the intestine of two gilthead seabream (Sparus aurata) homologs of mammalian aquaporin-1 (AQP1), named SaAqp1a and SaAqp1b. Heterologous expression in Xenopus laevis oocytes showed that SaAqp1a and SaAqp1b were water-selective channels. Real-time quantitative RT-PCR and Western blot using specific antisera indicated that abundance of SaAqp1a mRNA and protein was higher in duodenum and hindgut than in the rectum, whereas abundance of SaAqp1b was higher in rectum. In duodenum and hindgut, SaAqp1a localized at the apical brush border and lateral membrane of columnar enterocytes, whereas SaAqp1b was detected occasionally and at very low levels at the apical membrane. In the rectum, however, SaAqp1a was mainly accumulated in the cytoplasm of a subpopulation of enterocytes spread in groups over the surface of the epithelia, including the intervillus pockets, whereas SaAqp1b was detected exclusively at the apical brush border of all rectal enterocytes. Freshwater acclimation reduced the synthesis of SaAqp1a protein in all intestinal segments, but it only reduced SaAqp1b abundance in the rectum. These results show for the first time in teleosts a differential distribution and regulation of two functional AQP1 homologs in the intestinal epithelium, which suggest that they may play specialized functions during water movement across the intestine

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable -galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for -linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable -galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.

The gene encoding phenylacetyl-CoA ligase (pcl), the first enzyme of the pathway involved in the aerobic catabolism of phenylacetic acid in Pseudomonas putida U, has been cloned, sequenced, and expressed in two different microbes. In both, the primary structure of the protein was studied, and after genetic manipulation, different recombinant proteins were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresponding to the phenylacetyl-CoA ligase previously purified by us (Martínez-Blanco, H., Reglero, A. Rodríguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene in Escherichia coli leads to the appearance of this enzymatic activity, and cloning and expression of a 10.5-kb DNA fragment containing this gene confer this bacterium with the ability to grow in chemically defined medium containing phenylacetic acid as the sole carbon source. The appearance of phenylacetyl-CoA ligase activity in all of the strains of the fungus Penicillium chrysogenum transformed with a construction bearing this gene was directly related to a significant increase in the quantities of benzylpenicillin accumulated in the broths (between 1.8- and 2.2-fold higher), indicating that expression of this bacterial gene (pcl) helps to increase the pool of a direct biosynthetic precursor, phenylacetyl-CoA. This report describes the sequence of a phenylacetyl-CoA ligase for the first time and provides direct evidence that the expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for improving the biosynthetic machinery of this fungus.

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR). We used reverse transcription-polymerase chain reaction (RT-PCR) to obtain a partial pro-ARIA cDNA clone from methonine-1 to serine-358 including the full functional sequence of ARIA. Northern blot analysis of mRNAs from the embryonic chick brain and muscle showed a transcript with a size of similar to 7.5 kb. The cloned cDNA was subcloned into an eukaryotic expression vector and stably transfected into human embryonic kidney 293 cells. The conditioned medium of the transfected cells was found to increase the level of transcript encoding for the alpha-subunit of AChR by similar to 4.4-fold, but not for acetylcholinesterase (AChE), in the cultured chick myotubes.

The TRPV1 receptor plays a significant role in many biological processes, such as perception of external temperature (above 43°C), inflammation development, and thermoregulation. Activation of TRPV1 leads to the pain occurrence and decrease in the body temperature, while inhibition of this receptor can lead to an increase in the temperature. The TRPV1 peptide modulators from sea anemone Heteractis crispa extract (APHC1 and APHC3) have been previously characterized as molecules, which generated a pronounced analgesic effect and a decrease in the body temperature in experimental animals. Using the combined APHC1 and APHC3 amino acid sequences, we have prepared a hybrid peptide molecule named A13 that contains all residues potentially important for the activity of the peptide precursors. Biological tests on animals have shown that the hybrid molecule not only combines the analgesic properties of both peptides but, unlike the peptide precursors, also raises the body temperature of experimental animals. © 2017, Pleiades Publishing, Ltd.