Laccase: Sustainable production strategies, heterologous expression and potential biotechnological applications.

Laccase is a multicopper oxidase enzyme that target different types of phenols and aromatic amines. The enzyme can be isolated and characterized from microbes, plants and insects. Its ubiquitous nature and delignification ability makes it a valuable tool for research and development. Sustainable production methods are being employed to develop low cost biomanufacturing of the enzyme while achieving high titers. Laccase have significant industrial application ranging from food industry where it can be used for wine stabilization, texture improvement and detection of phenolic compounds in food products, to cosmetics offering benefits such as skin brightening and hair colouring. Dye decolourization/degradation, removal of pharmaceutical products/emerging pollutants and hydrocarbons from wastewater, biobleaching of textile fabrics, biofuel production and delignification of biomass making laccase a promising green biocatalyst. Innovative methods such as using inducers, microbial co-culturing, recombinant DNA technology, protein engineering have pivotal role in developing laccase with tailored properties. Enzyme immobilization using new age compounds including nanoparticles, carbonaceous components, agro-industrial residues enhance activity, stability and reusability. Commercial formulations of laccase have been prepared and readily available for a variety of applications. Certain challenges including production cost, metabolic stress in response to heterologous expression, difficulty in purification needs to be addressed.

Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in Streptococcus thermophilus.

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

Discovery and biosynthesis of non-canonical C16-terpenoids from Pseudomonas.

Biosynthesis of sodorifen with a unique C16-bicyclo[3.2.1]octene framework requires an S-adenosyl methionine-dependent methyltransferase SodC and terpene cyclase SodD. While bioinformatic analyses reveal a wide distribution of the sodCD genes organization in bacteria, their functional diversity remains largely unknown. Herein, two sodorifen-type gene clusters, pcch and pcau, from Pseudomonas sp. are heterologously expressed in Escherichia coli, leading to the discovery of two C16 terpenoids. Enzymatic synthesis of these compounds is achieved using the two (SodCD-like) pathway-specific enzymes. Enzyme assays using different combinations of methyltransferases and terpene synthases across the pcch, pcau, and sod pathways reveal a unifying biosynthetic mechanism: all three SodC-like enzymes methylate farnesyl pyrophosphate (FPP) with subsequent cyclization to a common intermediate, pre-sodorifen pyrophosphate. Structural diversification of this joint precursor solely occurs by the subsequently acting individual terpene synthases. Our findings expand basic biosynthetic understanding and structural diversity of unusual C16-terpenoids.

In vivo assembly in tobacco cells to elucidate and engineer the biosynthesis of 4-hydroxydihydrocinnamaldehyde from Gloriosa superba.

KEY MESSAGE: This study described the biosynthesis of 4-hydroxydihydrocinnamaldehyde sharing with monolignol pathway and supplemented the biosynthesis of colchicine in G. superba, 4-hydroxydihydrocinnamaldehyde produced in tobacco BY2 cells provided an important stepstone. The precursor, 4-hydroxydihydrocinnamaldehyde (4-HDCA), participates in the biosynthesis of the carbon skeleton of colchicine, which is derived from L-phenylalanine. However, one hypothesis proposed that 4-HDCA is synthesized by sharing the early part of the monolignol pathway in G. superba. In this study, we validated this prediction and identified the enzymatic functions involved in this pathway. GsDBR1 is a crucial enzyme to illustrate 4-HDCA diverging from monolignol pathway, we first confirmed its reductase activity on 4-coumaraldehyde, an important intermediate compound in monolignol biosynthesis. Then, the biochemical function of recombinant enzymes belonging to the other four families were verified to elucidate the entire process of 4-HDCA biosynthesis from L-phenylalanine. After reconstruction, the 4-HDCA was 78.4 ng/g with fresh weight (FW) of transgenic tobacco cells, and the yield increased to 168.22 ng/g·FW after improved treatment with methyl jasmonate (MeJA). The elucidation of 4-HDCA biosynthesis sharing the monolignol pathway supplemented the biosynthesis of colchicine in G. superba, and the production of 4-HDCA in tobacco cells provides an important step in the development of plant cell cultures as heterologous bio-factories for secondary metabolite production.

Heterologous immunization targeting the CST1 antigen confers better protection than ROP18 in mice.

Aim: To evaluate the protective efficacy induced by heterologous immunization with recombinant baculoviruses or virus-like particles targeting the CST1 and ROP18 antigens of Toxoplasma gondii.Materials & methods: Recombinant baculovirus and virus-like particle vaccines expressing T. gondii CST1 or ROP18 antigens were developed to evaluate protective immunity in mice upon challenge infection with 450 Toxoplasma gondii (ME49).Results: Immunization with CST1 or ROP18 vaccines induced similar levels of T. gondii-specific IgG and IgA responses. Compared with ROP 18, CST1 vaccine showed better antibody-secreting cell response, germinal center B cell activation, and significantly reduced brain cyst burden and body weight loss.Conclusion: Our findings suggest that CST1 heterologous immunization elicited better protection than ROP18, providing important insight into improving the toxoplasmosis vaccine design strategy.

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Screening and heterologous expression of an antimicrobial peptide SCAK33 with broad-spectrum antimicrobial activity resourced from sea cucumber proteome.

Antimicrobial peptides (AMPs) are a family of short defense proteins that are naturally produced by all organisms and have great potential as effective substitutes for small-molecule antibiotics. The present study aims to excavate AMPs from sea cucumbers and achieve their heterologous expression in prokaryotic Escherichia coli. Using MytC as a probe, a cysteine-stabilized peptide SCAK33 with broad-spectrum antimicrobial activity was discovered from the proteome of Apostichopus japonicas. The SCAK33 showed inhibitory effects on both gram positive and gram negative bacteria with MICs of 3-28 µM, and without significant hemolysis activity in rat blood erythrocyte. Especially, it exhibited good antimicrobial activity against Bacillus megaterium, B. subtilis, and Vibrio parahaemolyticus with the MIC of 3, 7, and 7 µM, respectively. After observation by scanning electronic microscopy (SEM) and confocal laser scanning microscope (CLSM), it was found that the cell membrane of bacteria was severely damaged. Furthermore, the recombinant SCAK33 (reSCAK33) was heterologously expressed by fusion with SUMO tag in E. coli BL21(DE3), and the protein yield reached 70 mg/L. The research will supplement the existing quantity of sea cucumber AMPs and provide data support for rapid mining and biological preparation of sea cucumber AMPs.

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures.

The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri. In pursuing our goal to capture each organism's population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture's health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE: Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, "state-of-the-art" labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum, C. ljungdahlii, and Clostridium kluyveri. We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

2-Ethylhexyl Diphenyl Phosphate Induces Autism Spectrum Disorder-Like Behaviors in Offspring Mice by Disrupting Postsynaptic Development.

As organophosphorus flame retardants (OPFRs) are constantly detected in human samples, the neurotoxicity of OPFRs is of concern. In this study, pregnant ICR mice were exposed to 2-ethylhexyl diphenyl phosphate (EHDPP) in drinking water from gestation to lactation to investigate its effects on autism spectrum disorder-like (ASD-like) behaviors in offspring. Serum EHDPP concentrations in dams in the 0.4, 2, and 10 mg/kg groups were 0.282 ± 0.051, 0.713 ± 0.115, and 0.974 ± 0.048 ng/mL, respectively, within the concentration range in humans. At the highest dose, EHDPP exposure induced ASD-like behaviors in both female and male offspring. Significant reductions in mature dendritic spines and structural damage to the postsynaptic density zone were noted in all but the lowest exposure groups, indicating postsynaptic membrane impairment. Mechanistically, EHDPP significantly downregulated disc large MAGUK scaffold protein 4 expression by inhibiting protein kinase B and type 1 insulin-like growth factor receptor phosphorylation. In the heterologous synapse formation assay in vivo, EHDPP significantly reduced the levels of postsynaptic density protein 95 expression in neurons at 1 µM. Overall, the study utilized in vitro and in vivo experiments to confirm that EHDPP damaged postsynaptic membrane formation and might increase the incidence of ASD in offspring.

Heterologous expression of the maize transcription factor ZmbHLH36 enhances abiotic stress tolerance in Arabidopsis.

Basic helix-loop-helix (bHLH) transcription factors are widely distributed in eukaryotes, and in plants, they regulate many biological processes, such as cell differentiation, development, metabolism, and stress responses. Few studies have focused on the roles of bHLH transcription factors in regulating growth, development, and stress responses in maize (Zea mays), even though such information would greatly benefit maize breeding programs. In this study, we cloned the maize transcription factor gene ZmbHLH36 (Gene ID: 100193615, GRMZM2G008691). ZmbHLH36 possesses conserved domains characteristic of the bHLH family. RT-qPCR analysis revealed that ZmbHLH36 was expressed at the highest level in maize roots and exhibited different expression patterns under various abiotic stress conditions. Transgenic Arabidopsis (Arabidopsis thaliana) plants heterologously expressing ZmbHLH36 had significantly longer roots than the corresponding non-transgenic plants under 0.1 and 0.15 mol L-1 NaCl treatment as well as 0.2 mol L-1 mannitol treatment. Phenotypic analysis of soil-grown plants under stress showed that transgenic Arabidopsis plants harboring ZmbHLH36 exhibited significantly enhanced drought tolerance and salt tolerance compared to the corresponding non-transgenic plants. Malondialdehyde contents were lower and peroxidase activity was higher in ZmbHLH36-expressing Arabidopsis plants than in the corresponding non-transgenic plants. ZmbHLH36 localized to the nucleus when expressed in maize protoplasts. This study provides a systematic analysis of the effects of ZmbHLH36 on root growth, development, and stress responses in transgenic Arabidopsis, laying a foundation for further analysis of its roles and molecular mechanisms in maize. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00159-3.

Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif.

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L-1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8-25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

Genetic Manipulation of Neisseria gonorrhoeae and Commensal Neisseria Species.

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

A novel thermophilic lysozyme 4356 from Cohnella sp. A01: Cloning, heterologous expression, biochemical and kinetic characterization.

Lysozymes have gained attention for their antiseptic properties. In silico studies have shown that the enzyme containing lysM can act as an antibacterial agent. Binding of the lysM motif of rSELys to peptidoglycan and molecular dynamics simulations showed that the protein-ligand binding is very stable. rSELys (2016 bp) is a new recombinant glycoside hydrolase from the thermophilic bacterium Cohnella sp. A01 (PTCC number: 1921). Protein expression and purification, a single band with an apparent molecular weight of ∼74 kDa was observed by SDS-PAGE. The kinetic parameters were Km 1.163 mg/ml, Vmax 670.3 U/mg, kcat 1675.75 (S-1), and kcat/Km 1440.88 (M-1S-1). Its optimum temperature was 55 °C and pH 8. Temperature stability also showed that the temperature of 50-60 °C retained more than half of its activity after 90 min. Based on the results, rSELys demonstrated antibacterial effects on both Gram-positive and Gram-negative strains, with inhibition zones of 11 and 9 mm, respectively. SEM analysis confirmed hydrolysis activity, the MIC was determined to be 31.25 µg/ml and 3.9 µg/ml, and MBC 0.97 µg/ml, respectively. CD and fluorescence studies showed that up to a temperature of 85 °C and a pH value of 8-12 no structural changes occur, and thermal stability protein was confirmed.

Advancements in the Heterologous Expression of Sucrose Phosphorylase and Its Molecular Modification for the Synthesis of Glycosylated Products.

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.

Age, Sex, and Training Specific Effects on Cross-Education Training.

An extensive number of publications have examined cross-education effects with adults, primarily investigating contralateral homologous (same) muscles. There are far fewer investigations on cross-education effects on contralateral heterologous (different) muscles and age (youth vs adult) and no studies investigating sex differences. Hence, the objective was to compare cross-education in female and male youth and young adults to contralateral homologous (chest press [CP], elbow flexors and extensors, handgrip isometric strength, and shot put) and heterologous (leg press, knee extension isometric strength, and countermovement jump) muscles. Twenty-eight female adults, 28 female youth, 28 male adults, and 28 male youth (total: 112) were examined before and after an 8-week (3 sessions/wk) unilateral, dominant arm, CP training program. Unilateral testing assessed dominant and nondominant leg press and CP 1-repetition maximum, knee extensors, elbow extensors, elbow flexors, and handgrip maximum voluntary isometric contraction (MVIC) strength, as well as shot put distance and countermovement jump height. Unilateral CP training induced training specific (CP 1-repetition maximum) and nonspecific (elbow extensors, elbow flexors, handgrip MVIC force, and shot put distance) improvements (P < .04, η2: .45-.85) but no significant lower body improvements. There was evidence for testing limb specificity as the dominant arm provided significantly (P < .021, η2: .17-.75) greater training gains than the nondominant arm. Youth's training adaptations exceeded with unilateral CP 1-repetition maximum, elbow extensors MVIC force, and shot put distance (P < .049, η2: .14-.49). No sex main effect differences were apparent. In conclusion, cross-education was training specific (greatest gains with upper body and dominant limbs) with greater benefits for youth and generally no sex differences with the exception of elbow extensors MVIC.

Dual-assist heterologous antigens screening: An effective strategy to improve the sensitivity of lateral flow immunoassay.

Heterologous strategy has promising applications in improving the sensitivity of competitive immunoassay. In this study, the potential heterologous coating antigens (HEA) were screened from eight imidacloprid (IMI) structural analogs based on the cross-reactivity (CR) of a prepared antibody. Computer-aided molecular modeling was used to predict the optimal HEA. Compared with the homologous coating antigen (HOA), the predicted HEA prepared from acetamiprid (CR = 0.23 %) increased the detection sensitivity of the enzyme-linked immunoassay and colloidal gold nanoparticle-based lateral flow immunoassay (HOA-Au-LFIA) by 5.6 and 4.1 times, respectively. Subsequently, the HEA and aggregation-induced emission fluorescent labels were integrated into a lateral flow immunoassay platform (HEA-AIE-LFIA). The limit of detection was 0.12 ng mL-1 for HEA-AIE-LFIA, which was 7.7-fold lower than that of HOA-Au-LFIA. Furthermore, HEA-AIE-LFIA was applied to detect IMI in food samples with excellent recoveries (86.41 %-111.25 %). Overall, this strategy of screening for superior HEA has great potential for improving LFIA sensitivity.

Expression of a novel hydrolase MhpC in Brevibacillus parabrevis BCP-09 and its characteristics for degrading synthetic pyrethroids.

Synthetic pyrethroids are widely used insecticides which may cause chronic diseases in non-target organisms upon long-term exposure. Microbial degradation offers a reliable method to remove them from the environment. This study focused on Brevibacillus parabrevis BCP-09 and its enzymes for degrading pyrethroids. The predicted deltamethrin-degrading genes phnA and mhpC were used to construct recombinant plasmids. These plasmids, introduced into Escherichia coli BL21(DE3) cells and induced with L-arabinose. The results indicated that the intracellular crude enzyme efficiently degraded deltamethrin by 98.8 %, ß-cypermethrin by 94.84 %, and cyfluthrin by 73.52 % within 24 h. The hydrolytic enzyme MhpC possesses a catalytic triad Ser/His/Asp and a typical "Gly-X-Ser-X-Gly" conservative sequence of the esterase family. Co-cultivation of induced E. coli PhnA and E. coli MhpC resulted in degradation rates of 41.44 ± 3.55 % and 60.30 ± 4.55 %, respectively, for deltamethrin after 7 d. This study states that the degrading enzymes from B. parabrevis BCP-09 are an effective method for the degradation of pyrethroids, providing available enzyme resources for food safety and environmental protection.

Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A2 inhibitor.

Crotoxin, a phospholipase A2 (PLA2) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA2 inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (~41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73%) CB-Cdc catalytic activity (5 moles), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA2s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA2 (5 moles) and reduced the CTx-Cdc (5 moles) enzyme activity by 65%, suppressing basic and acidic snake venom PLA2s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors.

An Interplay between Transcription Factors and Recombinant Protein Synthesis in Yarrowia lipolytica at Transcriptional and Functional Levels-The Global View.

Transcriptional regulatory networks (TRNs) associated with recombinant protein (rProt) synthesis in Yarrowia lipolytica are still under-described. Yet, it is foreseen that skillful manipulation with TRNs would enable global fine-tuning of the host strain's metabolism towards a high-level-producing phenotype. Our previous studies investigated the transcriptomes of Y. lipolytica strains overproducing biochemically different rProts and the functional impact of transcription factors (TFs) overexpression (OE) on rProt synthesis capacity in this species. Hence, much knowledge has been accumulated and deposited in public repositories. In this study, we combined both biological datasets and enriched them with further experimental data to investigate an interplay between TFs and rProts synthesis in Y. lipolytica at transcriptional and functional levels. Technically, the RNAseq datasets were extracted and re-analyzed for the TFs' expression profiles. Of the 140 TFs in Y. lipolytica, 87 TF-encoding genes were significantly deregulated in at least one of the strains. The expression profiles were juxtaposed against the rProt amounts from 125 strains co-overexpressing TF and rProt. In addition, several strains bearing knock-outs (KOs) in the TF loci were analyzed to get more insight into their actual involvement in rProt synthesis. Different profiles of the TFs' transcriptional deregulation and the impact of their OE or KO on rProts synthesis were observed, and new engineering targets were pointed.