The production of laccases by white-rot fungi under solid-state fermentation conditions.

Laccases (E.C. 1.10.3.2) produced by white-rot fungi (WRF) can be widely used, but the high cost prevents their use in large-scale industrial processes. Finding a solution to the problem could involve laccase production by solid-state fermentation (SSF) simulating the natural growth conditions for WRF. SSF offers several advantages over conventional submerged fermentation (SmF), such as higher efficiency and productivity of the process and pollution reduction. The aim of this review is therefore to provide an overview of the current state of knowledge about the laccase production by WRF under SSF conditions. The focus is on variations in the up-stream process, fermentation and down-stream process and their impact on laccase activity. The variations of up-stream processing involve inoculum preparation, inoculation of the medium and formulation of the propagation and production media. According to the studies, the production process can be shortened to 5-7 days by the selection of a suitable combination of lignocellulosic material and laccase producer without the need for any additional components of the culture medium. Efficient laccase production was achieved by valorisation of wastes as agro-food, municipal wastes or waste generated from wood processing industries. This leads to a reduction of costs and an increase in competitiveness compared to other commonly used methods and/or procedures. There will be significant challenges and opportunities in the future, where SSF could become more efficient and bring the enzyme production to a higher level, especially in new biorefineries, bioreactors and biomolecular/genetic engineering.

Expression Analysis of Lanosterol Synthase Gene in Dynamic Accumulation of Triterpenoids in Sanghuangporus baumii.

BACKGROUND: Sanghuangporus baumii is a traditional Chinese medicine with anti- cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear. OBJECTIVE: Some studies have shown that Lanosterol synthase ( LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis. METHODS: The PCR, RACE PCR, RT-PCR, homologous recombination and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level. RESULTS: The S. baumii LS sequence included a 5'-untranslated region (129 bp), a 3'-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased. CONCLUSION: The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.

Glyceric Prodrug of Ursodeoxycholic Acid (UDCA): Novozym 435-Catalyzed Synthesis of UDCA-Monoglyceride.

Bile acids (BAs) are a family of steroids synthesized from cholesterol in the liver. Among bile acids, ursodeoxycholic acid (UDCA) is the drug of choice for treating primary biliary cirrhosis and dissolving cholesterol gallstones. The clinical effectiveness of UDCA includes its choleretic activity, the capability to inhibit hydrophobic bile acid absorption by the intestine under cholestatic conditions, reducing cholangiocyte injury, stimulation of impaired biliary output, and inhibition of hepatocyte apoptosis. Despite its clinical effectiveness, UDCA is poorly soluble in the gastro-duodeno-jejunal contents, and pharmacological doses of UDCA are not readily soluble in the stomach and intestine, resulting in incomplete absorption. Indeed, the solubility of 20 mg/L greatly limits the bioavailability of UDCA. Since the bioavailability of drug products plays a critical role in the design of oral administration dosages, we investigated the enzymatic esterification of UDCA as a strategy of hydrophilization. Therefore, we decided to enzymatically synthesize a glyceric ester of UDCA bile acid to produce a more water-soluble molecule. The esterification reactions between UDCA and glycerol were performed with an immobilized lipase B from Candida antarctica (Novozym 435) in solvent-free and solvent-assisted systems. The characterization of the UDCA-monoglyceride, enzymatically synthesized, has been performed by 1H-NMR, 13C-NMR, COSY, HSQC, HMBC, IR, and MS spectroscopy.

Identifying carbohydrate-active enzymes of Cutaneotrichosporon oleaginosus using systems biology.

BACKGROUND: The oleaginous yeast Cutaneotrichosporon oleaginosus represents one of the most promising microbial platforms for resource-efficient and scalable lipid production, with the capacity to accept a wide range of carbohydrates encapsulated in complex biomass waste or lignocellulosic hydrolysates. Currently, data related to molecular aspects of the metabolic utilisation of oligomeric carbohydrates are sparse. In addition, comprehensive proteomic information for C. oleaginosus focusing on carbohydrate metabolism is not available. RESULTS: In this study, we conducted a systematic analysis of carbohydrate intake and utilisation by C. oleaginosus and investigated the influence of different di- and trisaccharide as carbon sources. Changes in the cellular growth and morphology could be observed, depending on the selected carbon source. The greatest changes in morphology were observed in media containing trehalose. A comprehensive proteomic analysis of secreted, cell wall-associated, and cytoplasmatic proteins was performed, which highlighted differences in the composition and quantity of secreted proteins, when grown on different disaccharides. Based on the proteomic data, we performed a relative quantitative analysis of the identified proteins (using glucose as the reference carbon source) and observed carbohydrate-specific protein distributions. When using cellobiose or lactose as the carbon source, we detected three- and five-fold higher diversity in terms of the respective hydrolases released. Furthermore, the analysis of the secreted enzymes enabled identification of the motif with the consensus sequence LALL[LA]L[LA][LA]AAAAAAA as a potential signal peptide. CONCLUSIONS: Relative quantification of spectral intensities from crude proteomic datasets enabled the identification of new enzymes and provided new insights into protein secretion, as well as the molecular mechanisms of carbo-hydrolases involved in the cleavage of the selected carbon oligomers. These insights can help unlock new substrate sources for C. oleaginosus, such as low-cost by-products containing difficult to utilize carbohydrates. In addition, information regarding the carbo-hydrolytic potential of C. oleaginosus facilitates a more precise engineering approach when using targeted genetic approaches. This information could be used to find new and more cost-effective carbon sources for microbial lipid production by the oleaginous yeast C. oleaginosus.

The Carbon Source Controls the Secretion and Yield of Polysaccharide-Hydrolyzing Enzymes of Basidiomycetes.

In the present study, the polysaccharide-hydrolyzing secretomes of Irpex lacteus (Fr.) Fr. (1828) BCC104, Pycnoporus coccineus (Fr.) Bondartsev and Singer (1941) BCC310, and Schizophyllum commune Fr. (1815) BCC632 were analyzed in submerged fermentation conditions to elucidate the effect of chemically and structurally different carbon sources on the expression of cellulases and xylanase. Among polymeric substrates, crystalline cellulose appeared to be the best carbon source providing the highest endoglucanase, total cellulase, and xylanase activities. Mandarin pomace as a growth substrate for S. commune allowed to achieve comparatively high volumetric activities of all target enzymes while wheat straw induced a significant secretion of cellulase and xylanase activities of I. lacteus and P. coccineus. An additive effect on the secretion of cellulases and xylanases by the tested fungi was observed when crystalline cellulose was combined with mandarin pomace. In I. lacteus the cellulase and xylanase production is inducible in the presence of cellulose-rich substrates but is suppressed in the presence of an excess of easily metabolizable carbon source. These enzymes are expressed in a coordinated manner under all conditions studied. It was shown that the substitution of glucose in the inoculum medium with Avicel provides accelerated enzyme production by I. lacteus and higher cellulase and xylanase activities of the fungus. These results add new knowledge to the physiology of basidiomycetes to improve cellulase production.

Structural Basis of Salicylic Acid Decarboxylase Reveals a Unique Substrate Recognition Mode and Access Channel.

Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M-1 s-1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M-1 s-1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.

The Simple Method of Preparation of Highly Carboxylated Bacterial Cellulose with Ni- and Mg-Ferrite-Based Versatile Magnetic Carrier for Enzyme Immobilization.

The bacterial cellulose (BC) is a versatile biopolymer of microbial origin characterized by high purity and unusual water and material properties. However, the native BC contains a low number of functional groups, which significantly limits its further application. The main goal of its effective modification is to use methods that allow the unusual properties of BC to be retained and the desired functional group to be efficiently introduced. In the present study, the new magnetic carrier based on functionalized citric acid (CA) bacterial cellulose was developed and tested to support critical industrial enzymes such as lipase B from Candida antarctica and phospholipase A from Aspergillus oryzae. The applied method allowed BC to be effectively modified by citric acid and a sufficient number of carboxylic groups to be introduced, up to 3.6 mmol of COOH per gram of dry mass of the prepared carrier. The DSC and TGA analyses revealed carrier stability at operational temperatures in the range of 20 °C to 100 °C and substantially influenced the amount of the introduced carboxyl groups on carrier properties. Both enzymes' immobilization significantly improves their thermal stability at 60 °C without a significant thermal and pH optima effect. The analyzed enzymes showed good operational stability with a significant residual activity after ten cycles of repeated uses. The new magnetic carrier based on highly carboxylated bacterial cellulose has a high application capability as matrix for immobilization the various enzymes of industrial interest.

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles.

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s' interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.

A Sesquiterpene Synthase from the Endophytic Fungus Serendipita indica Catalyzes Formation of Viridiflorol.

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.

Enhancing the thermostability of a mono- and diacylglycerol lipase from Malassizia globose by stabilizing a flexible loop in the catalytic pocket.

A lipase from Malassizia globose, named SMG1, is highly desirable for industrial application due to its substrate specificity towards mono- and diacylglycerol. To improve its thermostability, we constructed a mutant library using an error-prone polymerase chain reaction, which was screened for both initial and residual enzymatic activity. Selected mutants were further studied using purified proteins for their kinetic thermostability at 45 ℃, T50 (the temperature at which the enzyme loses half of its activity), and the optimal reaction temperature. Results showed that the majority of mutations with improved thermostability were on the protein surface. D245N and L270P showed the most significant thermostability enhancement with an approximately 3 ℃ increase in T50 compared to wild-type (WT). In addition, combining these two mutations resulted in an increase of T50 by 5 °C. Also, the optimal reaction temperatures of L270P and this double mutant are 10 ℃ higher than WT. The double mutant showed an approximately 100-fold increase in half-life at 45 ℃ and higher enzymatic activities at 30 ℃ and above compared to WT. High-temperature unfolding molecular dynamics simulation suggested that the double mutant stabilized a flexible loop in the catalytic pocket.

Effect of the acyl-group length on the chemoselectivity of the lipase-catalyzed acylation of propranolol-a computational study.

The selective N-acylation of 1,2-amino alcohols has been proposed to occur through the proton shuttle mechanism. However, the O-acetylation of propranolol catalyzed by Candida antarctica lipase B is an exception. We investigated the relation between the chemoselectivity of this reaction and the acyl group length. For this purpose, we compared the acyl groups: ethanoyl, butanoyl, octanoyl, and hexadecanoyl. We studied the Michaelis complexes between serine-acylated Candida antarctica lipase B and propranolol, employing a computational approach that involved sampling Michaelis complex conformations through ensemble docking plus consensus scoring and molecular dynamics simulations. The conformations were then classified as near attack conformations for acylation of the amino or hydroxy group. The relative populations of these two classes of conformations were found to be consistent with the experimentally observed chemoselective O-acetylation. We predict that increasing the length of the hydrocarbon chain of the acyl group will cause O-acylation to be unfavorable with respect to N-acylation. The nucleophilic attack of propranolol to the acylated lipase was found to be more favorable through the classical mechanism when compared with the proton shuttle mechanism.

Screening and characterization of a novel reversible 4-hydroxyisophthalic acid decarboxylase from Cystobasidium slooffiae HTK3.

Owing to carboxylation activity, reversible decarboxylases can use CO2 as a C1-building block to produce useful carboxylic acids. Although many reversible decarboxylases can synthesize aromatic monocarboxylic acids, only a few reversible decarboxylases have been reported to date that catalyze the synthesis of aromatic dicarboxylic acids. In the present study, a reversible 4-hydroxyisophthalic acid decarboxylase was identified in Cystobasidium slooffiae HTK3. Furthermore, recombinant 4-hydroxyisophthalic acid decarboxylase was prepared, characterized, and used for 4-hydroxyisophthalic acid production from 4-hydroxybenzoic acid.

A Bi-Enzymatic Cascade Pathway towards Optically Pure FAHFAs*.

Recently discovered endogenous mammalian lipids, fatty acid esters of hydroxy fatty acids (FAHFAs), have been proved to have anti-inflammatory and anti-diabetic effects. Due to their extremely low abundancies in vivo, forging a feasible scenario for FAHFA synthesis is critical for their use in uncovering biological mechanisms or in clinical trials. Here, we showcase a fully enzymatic approach, a novel in vitro bi-enzymatic cascade system, enabling an effective conversion of nature-abundant fatty acids into FAHFAs. Two hydratases from Lactobacillus acidophilus were used for converting unsaturated fatty acids to various enantiomeric hydroxy fatty acids, followed by esterification with another fatty acid catalyzed by Candida antarctica lipase A (CALA). Various FAHFAs were synthesized in a semi-preparative scale using this bi-enzymatic approach in a one-pot two-step operation mode. In all, we demonstrate that the hydratase-CALA system offers a promising route for the synthesis of optically pure structure-diverse FAHFAs.

Fibrinolytic and Thrombolytic Effects of an Enzyme Purified from the Fruiting Bodies of Boletus pseudocalopus (Agaricomycetes) from Korea.

A fibrinolytic enzyme with thrombolytic, anticoagulant activities was purified from fruiting bodies of wild-growing mushroom Boletus pseudocalopus Hongo and homogenized with a two-step procedure with a 6.11-fold increase in specific activity and 3.2% recovery. The molecular weight of the enzyme was estimated to be 63.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme was active at 40°C and pH 7, and activity was inhibited by zinc metal ion and by serine protease and trypsin inhibitors phenylmethylsulfonyl fluoride and N-α-tosyl-l-lysinyl-chloromethylketone. The enzyme displayed high specificity for Pyro-Glu-Gly-Arg-pNA. In vitro assays showed that the enzyme was able to degrade fibrin and blood clots, inhibit thrombin and activated factor X, and alter the density or structural change of fibrin clots. It could also delay activated partial thromboplastin time and prothrombin time. These results suggest that the enzyme may have characteristics of a trypsin or serine-like enzyme with fibrinolytic and thrombolytic activities and may have potential as an antithrombotic agent for blood clotting disorders.

Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies.

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

Evaluation of Different Ionic Liquids as Additives in the Immobilization of Lipase CAL B by Sol-Gel Technique.

Sol-gel technique aiming enzymatic immobilization in situ with ionic liquids as additives is poorly studied. In this process, the addition of the enzyme is carried out in the synthesis of the support. The characteristics of ionic liquids, such as low vapor pressure, thermal stability, and non-flammability, make them strong candidates for use as immobilization additives. The objective of the present study was to immobilize the Candida antarctica B lipase by the sol-gel technique using ionic liquids as additives. The optimum points determined for ionic liquids 1-butyl-3-methylimidazolium chloride, 1-octyl-3-methylimidazolium bromide, and 1 hexadecyl-3-methylimimidazolium were 0.30, 0.27, and 0.22 g/mL of enzyme and 1.60, 1.52, and 1.52% of additive, respectively. The amount of enzyme and ionic liquids used in aerogel immobilization was the same as the optimized values in the xerogel immobilization process (for each ionic liquid). Ionic liquids proved to be good additives in the enzymatic immobilization process. Xerogel, regardless of the ionic liquid, presented a greater number of use cycles and better thermal stability compared to aerogel.

Chitosan and Chitin Deacetylase Activity Are Necessary for Development and Virulence of Ustilago maydis.

The biotrophic fungus Ustilago maydis harbors a chitin deacetylase (CDA) family of six active genes as well as one pseudogene which are differentially expressed during colonization. This includes one secreted soluble CDA (Cda4) and five putatively glycosylphosphatidylinositol (GPI)-anchored CDAs, of which Cda7 belongs to a new class of fungal CDAs. Here, we provide a comprehensive functional study of the entire family. While budding cells of U. maydis showed a discrete pattern of chitosan staining, biotrophic hyphae appeared surrounded by a chitosan layer. We purified all six active CDAs and show their activity on different chitin substrates. Single as well as multiple cda mutants were generated and revealed a virulence defect for mutants lacking cda7 We implicated cda4 in production of the chitosan layer surrounding biotrophic hyphae and demonstrated that the loss of this layer does not reduce virulence. By combining different cda mutations, we detected redundancy as well as specific functions for certain CDAs. Specifically, certain combinations of mutations significantly affected virulence concomitantly with reduced adherence, appressorium formation, penetration, and activation of plant defenses. Attempts to inactivate all seven cda genes simultaneously were unsuccessful, and induced depletion of cda2 in a background lacking the other six cda genes illustrated an essential role of chitosan for cell wall integrity.IMPORTANCE The basidiomycete Ustilago maydis causes smut disease in maize, causing substantial losses in world corn production. This nonobligate pathogen penetrates the plant cell wall with the help of appressoria and then establishes an extensive biotrophic interaction, where the hyphae are tightly encased by the plant plasma membrane. For successful invasion and development in plant tissue, recognition of conserved fungal cell wall components such as chitin by the plant immune system needs to be avoided or suppressed. One strategy to achieve this lies in the modification of chitin to chitosan by chitin deacetylases (CDAs). U. maydis has seven cda genes. This study reveals discrete as well as redundant contributions of these genes to virulence as well as to cell wall integrity. Unexpectedly, the inactivation of all seven genes is not tolerated, revealing an essential role of chitosan for viability.

Disruption of protease A and B orthologous genes in the basidiomycetous yeast Pseudozyma antarctica GB-4(0) yields a stable extracellular biodegradable plastic-degrading enzyme.

The yeast Pseudozyma antarctica (currently designated Moesziomyces antarcticus) secretes a xylose-induced biodegradable plastic-degrading enzyme (PaE). To suppress degradation of PaE during production and storage, we targeted the inhibition of proteolytic enzyme activity in P. antarctica. Proteases A and B act as upper regulators in the proteolytic network of the model yeast, Saccharomyces cerevisiae. We searched for orthologous genes encoding proteases A and B in the genome of P. antarctica GB-4(0) based on the predicted amino acid sequences. We found two gene candidates, PaPRO1 and PaPRO2, with conserved catalytically important domains and signal peptides indicative of vacuolar protease function. We then prepared gene-deletion mutants of strain GB-4(0), ΔPaPRO1 and ΔPaPRO2, and evaluated PaE stability in culture by immunoblotting analysis. Both mutants exhibited sufficient production of PaE without degradation fragments, while the parent strain exhibited the degradation fragments. Therefore, we concluded that the protease A and B orthologous genes are related to the degradation of PaE. To produce a large quantity of PaE, we made a PaPRO2 deletion mutant of a PaE-overexpression strain named XG8 by introducing a PaE high-production cassette into the strain GB-4(0). The ΔPaPRO2 mutant of XG8 was able to produce PaE without the degradation fragments during large-scale cultivation in a 3-L jar fermenter for 3 days at 30°C. After terminating the agitation, the PaE activity in the XG8 ΔPaPRO2 mutant culture was maintained for the subsequent 48 h incubation at 25°C regardless of remaining cells, while activity in the XG8 control was reduced to 55.1%. The gene-deleted mutants will be useful for the development of industrial processes of PaE production and storage.