Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris.

OBJECTIVES: Different cultivation conditions and parameters were evaluated to improve the production and secretion of a recombinant Phanerochaete chrysosporium lipH8 gene in Komagataella phaffii (Pichia pastoris). RESULTS: The recombinant lipH8 gene with its native secretion signal was successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) under the control of the alcohol oxidase 1 promoter (PAOX1). The results revealed that co-feeding with sorbitol and methanol increased rLiP secretion by 5.9-fold compared to the control conditions. The addition of 1 mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Moreover, the combination of several optimal conditions and parameters yielded an extracellular rLiP activity of 20.05 U l-1, which is more than ten-fold higher relative to standard growth conditions (BMM10 medium, pH 6 and 30 °C). CONCLUSION: Extracellular activity of a recombinant LiP expressed in P. pastoris increased more than ten-fold when co-feeding sorbitol and methanol as carbon sources, together with urea as nitrogen source, FeSO4 supplementation, lower pH and lower cultivation temperature.

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.

Tobacco Plastid Transformation as Production Platform of Lytic Polysaccharide MonoOxygenase Auxiliary Enzymes.

Plant biomass is the most abundant renewable resource in nature. In a circular economy perspective, the implementation of its bioconversion into fermentable sugars is of great relevance. Lytic Polysaccharide MonoOxygenases (LPMOs) are accessory enzymes able to break recalcitrant polysaccharides, boosting biomass conversion and subsequently reducing costs. Among them, auxiliary activity of family 9 (AA9) acts on cellulose in synergism with traditional cellulolytic enzymes. Here, we report for the first time, the production of the AA9 LPMOs from the mesophilic Trichoderma reesei (TrAA9B) and the thermophilic Thermoascus aurantiacus (TaAA9B) microorganisms in tobacco by plastid transformation with the aim to test this technology as cheap and sustainable manufacture platform. In order to optimize recombinant protein accumulation, two different N-terminal regulatory sequences were used: 5' untranslated region (5'-UTR) from T7g10 gene (DC41 and DC51 plants), and 5' translation control region (5'-TCR), containing the 5'-UTR and the first 14 amino acids (Downstream Box, DB) of the plastid atpB gene (DC40 and DC50 plants). Protein yields ranged between 0.5 and 5% of total soluble proteins (TSP). The phenotype was unaltered in all transplastomic plants, except for the DC50 line accumulating AA9 LPMO at the highest level, that showed retarded growth and a mild pale green phenotype. Oxidase activity was spectrophotometrically assayed and resulted higher for the recombinant proteins without the N-terminal fusion (DC41 and DC51), with a 3.9- and 3.4-fold increase compared to the fused proteins.

Expression of a ß-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Combinations of slow-translating codon clusters can increase mRNA half-life in Saccharomyces cerevisiae.

The presence of a single cluster of nonoptimal codons was found to decrease a transcript's half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae The impact of multiple nonoptimal codon clusters on a transcript's half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5' end can lead to synergistic effects that increase a messenger RNA's (mRNA's) half-life in S. cerevisiae Specifically, the 5' end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5' end is the primary determinant of mRNA half-life. These results suggest the "translational ramp," in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates.

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli.

BACKGROUND: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli. RESULTS: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused. CONCLUSIONS: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α-promoter. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

Increased Expression of Recombinant Chitosanase by Co-expression of Hac1p in the Yeast Pichia pastoris

BACKGROUND: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR). OBJECTIVE: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris. METHOD: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. RESULTS: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulting from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1 further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression withPAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. CONCLUSION: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.

Biosynthesis of a New Fusaoctaxin Virulence Factor in Fusarium graminearum Relies on a Distinct Path To Form a Guanidinoacetyl Starter Unit Priming Nonribosomal Octapeptidyl Assembly.

Fusarium graminearum is a pathogenic fungus causing huge economic losses worldwide via crop infection leading to yield reduction and grain contamination. The process through which the fungal invasion occurs remains poorly understood. We recently characterized fusaoctaxin A in F. graminearum, where this octapeptide virulence factor results from an assembly line encoded in fg3_54, a gene cluster proved to be involved in fungal pathogenicity and host adaptation. Focusing on genes in this cluster that are related to fungal invasiveness but not to the biosynthesis of fusaoctaxin A, we here report the identification and characterization of fusaoctaxin B, a new octapeptide virulence factor with comparable activity in wheat infection. Fusaoctaxin B differs from fusaoctaxin A at the N-terminus by possessing a guanidinoacetic acid (GAA) unit, formation of which depends on the combined activities of the protein products of fgm1-3. Fgm1 is a cytochrome P450 protein that oxygenates l-Arg to 4(R)-hydroxyl-l-Arg in a regio- and stereoselective manner. Then, Cß-Cγ bond cleavage proceeds in the presence of Fgm3, a pyridoxal-5'-phosphate-dependent lyase, giving guanidinoacetaldehyde and l-Ala. Rather than being directly oxidized to GAA, the guanidine-containing aldehyde undergoes spontaneous cyclization and subsequent enzymatic dehydrogenation to provide glycociamidine, which is linearized by Fgm2, a metallo-dependent amidohydrolase. The GAA path in F. graminearum is distinct from that previously known to involve l-Arg:l-Gly aminidotransferase activity. To provide this nonproteinogenic starter unit that primes nonribosomal octapeptidyl assembly, F. graminearum employs new chemistry to process l-Arg through inert C-H bond activation, selective C-C bond cleavage, cyclization-based alcohol dehydrogenation, and amidohydrolysis-associated linearization.

Protease Produced by Endophytic Fungi: A Systematic Review.

The purpose of this systematic review was to identify the available literature of production, purification, and characterization of proteases by endophytic fungi. There are few complete studies that entirely exhibit the production, characterization, and purification of proteases from endophytic fungi. This study followed the PRISMA, and the search was conducted on five databases: PubMed, PMC, Science Direct, Scopus Articles, and Web of Science up until 18 May 2021, with no time or language restrictions. The methodology of the selected studies was evaluated using GRADE. Protease production, optimization, purification, and characterization were the main evaluated outcomes. Of the 5540 initially gathered studies, 15 met the inclusion criteria after a two-step selection process. Only two studies optimized the protease production using statistical design and two reported enzyme purification and characterization. The genus Penicillium and Aspergillus were the most cited among the eleven different genera of endophytic fungi evaluated in the selected articles. Six studies proved the ability of some endophytic fungi to produce fibrinolytic proteases, demonstrating that endophytic fungi can be exploited for the further production of agents used in thrombolytic therapy. However, further characterization and physicochemical studies are required to evaluate the real potential of endophytic fungi as sources of industrial enzymes.

Carbon starvation-induced synthesis of GDH2 and PEPCK is essential for the survival of Pichia pastoris.

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.

Nosema bombycis microRNA-like RNA 8 (Nb-milR8) Increases Fungal Pathogenicity by Modulating BmPEX16 Gene Expression in Its Host, Bombyx mori.

The fungus Nosema bombycis causes significant economic losses via parasitism of an economically important insect. MicroRNAs (miRNAs) play important roles in regulating host and parasite gene expression via mRNA degradation or by inhibiting protein translation. To investigate whether microRNA-like RNAs (milRNAs) regulate N. bombycis pathogenesis and to better understand the regulatory mechanisms underlying infection, we constructed small RNA libraries from N. bombycis hyphae during the schizont proliferation period. Eleven novel milRNAs were determined by RNA sequencing and stem-loop reverse transcriptase PCR (RT-PCR) assays. Moreover, a virulence-associated milRNA, Nb-milR8, was identified as critical for N. bombycis proliferation by binding and downregulating expression of its target gene, BmPEX16, in the host during infection. Silencing of Nb-milR8 or overexpression of the target BmPEX16 gene resulted in increased susceptibility of Bombyx mori to N. bombycis infection. Taken together, these results suggest that Nb-milR8 is an important virulence factor that acts as an effector to suppress host peroxidase metabolism, thereby facilitating N. bombycis proliferation. These results provide important novel insights into interactions between pathogenic fungi and their hosts. IMPORTANCE A thorough understanding of fungal pathogen adaptations is essential for treating fungal infections. Recent studies have suggested that the role of small RNAs expressed in fungal microsporidia genomes are important for elucidating the mechanisms of fungal infections. Here, we report 11 novel microRNA-like RNAs (milRNAs) from the fungal microsporidium Nosema bombycis and identified NB-milRNAs that adaptively regulate N. bombycis proliferation. In addition, we demonstrate that N. bombycis modulates small RNA (sRNA)-mediated infection by encoding an Nb-miR8 that downregulates the expression of the host peroxidase metabolism protein BmPEX16, which is essential for peroxisome membrane biogenesis and peroxisome assembly. These results significantly contribute to our understanding of the pathogenic mechanisms of fungi, and especially microsporidia, while providing important targets for genetical engineering-based treatment of microsporidia.

Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

The Neurospora crassa Inducible Q System Enables Simultaneous Optogenetic Amplification and Inversion in Saccharomyces cerevisiae for Bidirectional Control of Gene Expression.

Bidirectional optogenetic control of yeast gene expression has great potential for biotechnological applications. Our group has developed optogenetic inverter circuits that activate transcription using darkness, as well as amplifier circuits that reach high expression levels under limited light. However, because both types of circuits harness Gal4p and Gal80p from the galactose (GAL) regulon they cannot be used simultaneously. Here, we apply the Q System, a transcriptional activator/inhibitor system from Neurospora crassa, to build circuits in Saccharomyces cerevisiae that are inducible using quinic acid, darkness, or blue light. We develop light-repressed OptoQ-INVRT circuits that initiate darkness-triggered transcription within an hour of induction, as well as light-activated OptoQ-AMP circuits that achieve up to 39-fold induction. The Q System does not exhibit crosstalk with the GAL regulon, allowing coutilization of OptoQ-AMP circuits with previously developed OptoINVRT circuits. As a demonstration of practical applications in metabolic engineering, we show how simultaneous use of these circuits can be used to dynamically control both growth and production to improve acetoin production, as well as enable light-tunable co-production of geraniol and linalool, two terpenoids implicated in the hoppy flavor of beer. OptoQ-AMP and OptoQ-INVRT circuits enable simultaneous optogenetic signal amplification and inversion, providing powerful additions to the yeast optogenetic toolkit.

Fungal phytochrome chromophore biosynthesis at mitochondria.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

Microbial production, molecular modification, and practical application of l-Asparaginase: A review.

L-Asparaginase (L-ASNase, EC 3.5.1.1), an antitumor drug for acute lymphoblastic leukemia (ALL) therapy, is widely used in the clinical field. Similarly, L-ASNase is also a powerful and significant biological tool in the food industry to inhibit acrylamide (AA) formation. This review comprehensively summarizes the latest achievements and improvements in the production, modification, and application of microbial L-ASNase. To date, the expression levels and optimization of expression hosts such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, have made significant progress. In addition, examples of successful modification of L-ASNase such as decreasing glutaminase activity, increasing the in vivo stability, and enhancing thermostability have been presented. Impressively, the application of L-ASNase as a food addition aid, as well as its commercialization in the pharmaceutical field, and cutting-edge biosensor application developments have been summarized. The presented results and proposed ideas could be a good guide for other L-ASNase researchers in both scientific and practical fields.

GhKWL1 Upregulates GhERF105 but Its Function Is Impaired by Binding with VdISC1, a Pathogenic Effector of Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five KWL genes (GhKWLs) were identified from the genome of upland cotton (Gossypium hirsutum). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of V. dahliae. We revealed that GhKWL1 was a positive regulator of GhERF105. Silencing of GhKWL1 resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by V. dahliae could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by V. dahliae through the interaction of VdISC1 with GhKWL1.

Albumin Neutralizes Hydrophobic Toxins and Modulates Candida albicans Pathogenicity.

Albumin is abundant in serum but is also excreted at mucosal surfaces and enters tissues when inflammation increases vascular permeability. Host-associated opportunistic pathogens encounter albumin during commensalism and when causing infections. Considering the ubiquitous presence of albumin, we investigated its role in the pathogenesis of infections with the model human fungal pathogen, Candida albicans. Albumin was introduced in various in vitro models that mimic different stages of systemic or mucosal candidiasis, where it reduced the ability of C. albicans to damage host cells. The amphipathic toxin candidalysin mediates necrotic host cell damage induced by C. albicans. Using cellular and biophysical assays, we determined that albumin functions by neutralizing candidalysin through hydrophobic interactions. We discovered that albumin, similarly, can neutralize a variety of fungal (α-amanitin), bacterial (streptolysin O and staurosporin), and insect (melittin) hydrophobic toxins. These data suggest albumin as a defense mechanism against toxins, which can play a role in the pathogenesis of microbial infections. IMPORTANCE Albumin is the most abundant serum protein in humans. During inflammation, serum albumin levels decrease drastically, and low albumin levels are associated with poor patient outcome. Thus, albumin may have specific functions during infection. Here, we describe the ability of albumin to neutralize hydrophobic microbial toxins. We show that albumin can protect against damage induced by the pathogenic yeast C. albicans by neutralizing its cytolytic toxin candidalysin. These findings suggest that albumin is a toxin-neutralizing protein that may play a role during infections with toxin-producing microorganisms.