Recombinant expression of fungal oxidases for industrial application

Laccases catalyse the oxidation of a range of organic substrates coupled to the reduction of molecular oxygen to water. They are members of the ubiquitous blue multi-copper oxidase family. These enzymes are implicated in a wide variety of biological activities. Most of the laccases studied thus far are of fungal origin. Large variety of potential substrates has raised interest in the use of laccases in several industrial applications, such as pulp delignification, textile dye bleaching, effluent detoxification, biopolymer modification and bioremediation. Cloning of the laccase genes followed by heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (different substrate specificities and improved stabilities) for industrial applications. Heterologous expression of Pleurotus ostreatus laccases POXC and POXA1b in two yeasts and a first approach of directed evolution experiments are reported. The yeasts of choice were Saccharomyces cerevisiae, proven to be success-full in recombinant laccase expression and directed-evolution experiments, and Kluyveromyces lactis, a non-conventional yeast offering significant advantages, such as high-level secretion of non-hyperglycosylated recombinant proteins. Expression vectors were set up cloning the cDNAs under the control of different promoters. Furthermore, the laccase leader peptides (poxc and poxa1b), as well as the yeast derived signal peptides (S. cerevisiae invertase and K. lactis killer toxin), were alternatively used to direct the secretion of active laccase into the culture medium. The laccase signals proved to be more effective to drive the secretion of recombinant proteins in both hosts. Levels of laccase secreted activity were markedly different: rPOXA1b transformants always gave much higher activity than rPOXC transformants, and production of both laccases in S. cerevisiae was significantly lower than that in K. lactis. Recombinant laccases from K. lactis were purified to electrophoretic homogeneity and characterized. rPOXA1b specific activity was similar to that of the native protein, whilst rPOXC specific activity was much lower than that of the native POXC. Mass spectrometry analyses of the recombinant proteins allowed to verify their primary structures and to identify post-translational modifications. Our data confirm that K. lactis has a lower tendency, respect to S. cerevisiae, to hyperglycosylate recombinant proteins. The S. cerevisiae laccase expression systems were further used to set off directed evolution experiments. Mutated cDNAs libraries with different mutation rate were created, and homologous recombination experiments were performed, giving rise to libraries of mutated laccase secreting yeasts. Moreover a screening procedure to isolate clones exhibiting desired property was realized. As a result, this work allowed obtaining the heterologous expression of two P. ostreatus laccases in yeasts, and their purification and characterisation. Moreover, this research work broadened the potentiality of the developed expression system addressing enzymes to such large markets and different industrial application such as pulp and textile bleaching, and enzymatic remediation of waste streams. A new laccase host (K. lactis) has been built on, and its promising performances will lead to further investigate its utilization for further structure-activities studies, as well as for directed evolution. Results obtained demonstrate the potential of the recombinant expression for the study of potential industrial interest

Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Induction and Transcriptional Regulation of Laccases in Fungi

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet

Diagnostic and therapeutic path of breast cancer: Effectiveness, appropriateness, and costs â Results from the DOCMa study

none8noopenGiovagnoli, Maria Rosaria; Bonifacino, Adriana; Neglia, Cosimo; Benvenuto, Marco; Sambati, Francesco Vincenzo; Giolli, Lorenzo; Giovagnoli, Alessandra; Piscitelli, PriscoGiovagnoli, Maria Rosaria; Bonifacino, Adriana; Neglia, Cosimo; Benvenuto, Marco; Sambati, Francesco Vincenzo; Giolli, Lorenzo; Giovagnoli, Alessandra; Piscitelli, Prisc

Healthy aging and cardiovascular health in Kyrgyzstan: current status and emerging challenges

s Kyrgyzstan undergoes significant demographic transitions, with an increasing aging population, the intersection of healthy aging and cardiovascular health emerges as a critical aspect of public health. We explore the relationship of healthy aging and cardiovascular health, underlining both the opportunities and challenges inherent in this demographic shift. Applying the World Health Organization's concept of "intrinsic capacity" and the "Integrated Care for Older People" (ICOPE) approach, we suggest strategies for addressing these crucial public health issues

Infection in a Geriatric Patient: Do Not Let Your Guard Off!

Tetanus is an infectious disease caused by Clostridium tetani toxin. Although easily preventable through vaccination, over 73,000 new infections and 35,000 deaths due to tetanus occurred worldwide in 2019, with higher rates in countries with healthcare barriers. Here, we present a clinical case of C. tetani infection in an 85-year-old patient. Patient robustness and high functional reserve before infection are favorable predictors of survival for an otherwise fatal disease. However, the patient did not experience any severe complications. Therefore, this report is a strong call for tetanus vaccination

Metabolic and microbiota response to arginine supplementation and cyclic heat stress in broiler chickens

Little attention has been paid to the biological role of arginine and its dietary supplementation in broilers under heat stress (HS) conditions. Therefore, the main aim of this study was to assess the response of broilers to arginine supplementation and cyclic HS, with a focus on liver, pectoral muscle, and blood metabolic profiles and the cecal microbiota. Day-old male Ross 308 broilers (n = 240) were placed in 2 rooms with 12 pens each for a 44-day trial. Pens were assigned to one of two groups (6 pens/group/room): the control group (CON) was given a basal diet in mash form and the treated group (ARG) was fed CON diet supplemented with crystalline L-arginine. The total arginine:lysine ratio of CON diet ranged between 1.02 and 1.07, while that of ARG diet was 1.20. One room was constantly kept at thermoneutral (TN) conditions, while the birds in the other room were kept at TN conditions until D34 and subjected to cyclic HS from D35 onwards (∼34°C; 9:00 A.M.–6:00 P.M.). Blood, liver, Pectoralis major muscle, and cecal content were taken from 2 birds per pen (12 birds/group/room) for metabolomics and microbiota analysis. Growth performance data were also collected on a pen basis. Arginine supplementation failed to reduce the adverse effects of HS on growth performance. Supplemented birds showed increased levels of arginine and creatine in plasma, liver, and P. major and methionine in liver, and reduced levels of glutamine in plasma, liver, and P. major. HS altered bioenergetic processes (increased levels of AMP and reduced levels of fumarate, succinate, and UDP), protein metabolism (increased protein breakdown to supply the liver with amino acids for energy production), and promoted the accumulation of antioxidant and protective molecules (histidine-containing dipeptides, beta-alanine, and choline), especially in P. major. Arginine supplementation may have partially counterbalanced the effects of HS on energy homeostasis by increasing creatine levels and attenuating the increase in AMP levels, particularly in P. major. It also significantly reduced cecal observed diversity, while HS increased alpha diversity indices and affected beta diversity. Results of taxonomic analysis at the phylum and family level are also provided

Toward highly potent cancer agents by modulating the c-2 group of the arylthioindole class of tubulin polymerization inhibitors

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds 18 and 57 were superior to the previously synthesized 5. Compound 18 was exceptionally potent as an inhibitor of cell growth: it showed IC50 = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds 18, 20, 55, and 57 were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds 18 and 57 showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes