Polarity-dependent expression and localization of secretory glucoamylase mRNA in filamentous fungal cells.

In multinuclear and multicellular filamentous fungi little is known about how mRNAs encoding secreted enzymes are transcribed and localized spatiotemporally. To better understand this process we analyzed mRNA encoding GlaA, a glucoamylase secreted in large amounts by the industrial filamentous fungus Aspergillus oryzae, by the MS2 system, in which mRNA can be visualized in living cells. We found that glaA mRNA was significantly transcribed and localized near the hyphal tip and septum, which are the sites of protein secretion, in polarity-dependent expression and localization manners. We also revealed that glaA mRNA exhibits long-range dynamics in the vicinity of the endoplasmic reticulum (ER) in a manner that is dependent on the microtubule motor proteins kinesin-1 and kinesin-3, but independent of early endosomes. Moreover, we elucidated that although glaA mRNA localized to stress granules (SGs) and processing bodies (PBs) under high temperature, glaA mRNA was not seen under ER stress, suggesting that there are different regulatory mechanisms of glaA mRNA by SG and PB under high temperature and ER stress. Collectively, this study uncovers a dynamic regulatory mechanism of mRNA encoding a secretory enzyme in filamentous fungi.

Development of the thermophilic fungus Myceliophthora thermophila into glucoamylase hyperproduction system via the metabolic engineering using improved AsCas12a variants.

BACKGROUND: Glucoamylase is an important enzyme for starch saccharification in the food and biofuel industries and mainly produced from mesophilic fungi such as Aspergillus and Rhizopus species. Enzymes produced from thermophilic fungi can save the fermentation energy and reduce costs as compared to the fermentation system using mesophiles. Thermophilic fungus Myceliophthora thermophila is industrially deployed fungus to produce enzymes and biobased chemicals from biomass during optimal growth at 45 °C. This study aimed to construct the M. thermophila platform for glucoamylase hyper-production by broadening genomic targeting range of the AsCas12a variants, identifying key candidate genes and strain engineering. RESULTS: In this study, to increase the genome targeting range, we upgraded the CRISPR-Cas12a-mediated technique by engineering two AsCas12a variants carrying the mutations S542R/K607R and S542R/K548V/N552R. Using the engineered AsCas12a variants, we deleted identified key factors involved in the glucoamylase expression and secretion in M. thermophila, including Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2. Deletion of four targets led to more than 1.87- and 1.85-fold higher levels of secretion and glucoamylases activity compared to wild-type strain MtWT. Transcript level of the major amylolytic genes showed significantly increased in deletion mutants. The glucoamylase hyper-production strain MtGM12 was generated from our previously strain MtYM6 via genetically engineering these targets Mtstk-12, Mtap3m, Mtdsc-1 and Mtsah-2 and overexpressing Mtamy1 and Mtpga3. Total secreted protein and activities of amylolytic enzymes in the MtGM12 were about 35.6-fold and 51.9‒55.5-fold higher than in MtWT. Transcriptional profiling analyses revealed that the amylolytic gene expression levels were significantly up-regulated in the MtGM12 than in MtWT. More interestingly, the MtGM12 showed predominantly short and highly bulging hyphae with proliferation of rough ER and abundant mitochondria, secretion vesicles and vacuoles when culturing on starch. CONCLUSIONS: Our results showed that these AsCas12a variants worked well for gene deletions in M. thermophila. We successfully constructed the glucoamylase hyper-production strain of M. thermophila by the rational redesigning and engineering the transcriptional regulatory and secretion pathway. This targeted engineering strategy will be very helpful to improve industrial fungal strains and promote the morphology engineering for enhanced enzyme production.

A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about 70 µg·mL-1 PatE protein in the culture medium with a purity just under 90%.

Enhanced expression of xylanase in Aspergillus niger enabling a two-step enzymatic pathway for extracting ß-glucan from oat bran.

The high cost and process complexity limit the enzymatic extraction of ß-glucan. In this study, ß-glucan was extracted from oat bran in a two-step enzymatic pathway using a recombinant strain of Aspergillus niger AG11 overexpressing the endogenous xylanase (xynA) and amylolytic enzyme. First, co-optimization of promoter and signal peptide and a fusion of glucoamylase (glaA) fragment were integrated into the ß-glucosidase (bgl) locus to improve xynA expression. Then, the optimized expression cassette was simultaneously integrated into bgl, α-amylase amyA, and acid α-amylase ammA loci, yielding the Rbya with 3,650-fold and 31.2% increase in xynA and amylolytic enzyme activity than the wild-type strain, respectively. Finally, Rbya's supernatants at 72 h (rich in xynA and amylolytic enzyme) and 10 d (rich in proteases) were used to decompose xylan/starch and proteins in oat bran, respectively, to obtain 85.1% pure ß-glucan. Rbya could be a robust candidate for the cost-effective extraction of ß-glucan.

Improving the Catalytic Efficiency of Aspergillus fumigatus Glucoamylase toward Raw Starch by Engineering Its N-Glycosylation Sites and Saturation Mutation.

Raw starch glucoamylase (RSGA) can degrade the raw starch below the starch gelatinization temperature. In this study, to improve the catalytic activity of raw corn starch, N-glycosylation was introduced into the RSGA from Aspergillus fumigatus through site-directed mutation and the recombinant expression in Komagataella phaffii. Among them, the mutants G101S (N99-L100-S101) and Q113T (N111-S112-T113) increased the specific activity of raw corn starch by 1.19- and 1.21-fold, respectively. The optimal temperature of Q113T decreased from 70 to 60 °C. Notably, the combined mutant G101S/Q113T increased the specific activity toward raw starch by 1.22-fold and reduced the optimal temperature from 70 to 60 °C. Moreover, the mutant Q113M with a 1.5-fold increase in the catalytic activity was obtained via saturation mutation at site 113. Thus, the N-glycosylation site engineering is an efficient method to improve the activity of RSGA toward raw starch.

[Assessment of the influence of an enzymal preparation - a complex of glucoamylase and xylanase from Aspergillus awamori Xyl T-15 on the intestinal microbiom and immunological indicators of rats].

The requirements for the safety of food products obtained by microbial synthesis are including as obligation for to conduct toxicological studies - the study of various biochemical and immunological markers of toxic effects. The necessity of these studies is explained by a possible change in the structure of food ingredients produced by a microbial cell and, consequently, a change in their biological properties, as well as the possible presence of living forms and/or DNA of producer strains or of their toxic metabolites in these ingredients. At the same time, it is well known that the nutrient composition of foods has a significant impact on the composition and properties of microorganisms that make up the gut microbiome, which, in turn, determines the immune status. The purpose of the research was to justify the analyses of gut microbiocenosis composition for inclusion in the protocol of safety investigation of foods obtained by microbial synthesis [on the example of an enzyme preparation (EP) - a complex of glucoamylase and xylanase from a genetically modified strain of Aspergillus awamori Xyl T-15]. Material and methods. In experimental studies carried out for 80 days, Wistar rats (males and females) were used. The study of the effect of EP (a complex of glucoamylase and xylanase from a genetically modified Aspergillus awamori Xyl T-15 strain) in dozes 10, 100 and 1000 mg/kg body mass on the cecum microbiome and the immune status (content of cytokines and chemokines: IL-1a, IL-4, IL-6, IL-10, IL-17A, INF-γ, TNF-α, MCP-1, MIP-1a and Regulated on Activation Normal T-cell Expressed and Secreted - RANTES) was carried out. Results. It has been shown that EP - a complex of glucoamylase and xylanase from A. awamori Xyl T-15 at doses of 100 mg/kg or more causes mild disturbances in the composition of gut microbiocenosis. At the same time, these disorders have a significant immunomodulat ory and immunotoxic effect on the body, which manifests itself in a dose-dependent change in the profile of pro-inflammatory cytokines and chemokines in blood and spleen. The adverse effect of EP on the body is probably due to the formation of metabolites that are not formed during usual digestive processes in the gastrointestinal tract. The minimum effective dose (LOAEL) of EP was 100 mg/kg body weight In accordance with established requirements, the activity of the EP should not appear in ready-to-use food. Subject to this requirement, amount of EP entering the body cannot exceed the established LOAEL level. Therefore, a complex of glucoamylase and xylanase can be used in food industry, subject to the establishment of regulations «for technological purposes¼ for A. awamori Xyl T-15 strain. Conclusion. The data obtained on the relationship between the state of the microbiome and the immune status upon the introduction of EP indicate the need to include indicators of the state of gut microbiocenosis in the test protocol of safety.

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger.

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi.

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

A cell engineering approach to enzyme-based fed-batch fermentation.

BACKGROUND: A fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation. RESULTS: A putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent. CONCLUSIONS: This study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation.

Muscle Proteomic Profile before and after Enzyme Replacement Therapy in Late-Onset Pompe Disease.

Mutations in the acidic alpha-glucosidase (GAA) coding gene cause Pompe disease. Late-onset Pompe disease (LOPD) is characterized by progressive proximal and axial muscle weakness and atrophy, causing respiratory failure. Enzyme replacement therapy (ERT), based on recombinant human GAA infusions, is the only available treatment; however, the efficacy of ERT is variable. Here we address the question whether proteins at variance in LOPD muscle of patients before and after 1 year of ERT, compared withhealthy age-matched subjects (CTR), reveal a specific signature. Proteins extracted from skeletal muscle of LOPD patients and CTR were analyzed by combining gel based (two-dimensional difference gel electrophoresis) and label-free (liquid chromatography-mass spectrometry) proteomic approaches, and ingenuity pathway analysis. Upstream regulators targeting autophagy and lysosomal tethering were assessed by immunoblotting. 178 proteins were changed in abundance in LOPD patients, 47 of them recovered normal level after ERT. Defects in oxidative metabolism, muscle contractile protein regulation, cytoskeletal rearrangement, and membrane reorganization persisted. Metabolic changes, ER stress and UPR (unfolded protein response) contribute to muscle proteostasis dysregulation with active membrane remodeling (high levels of LC3BII/LC3BI) and accumulation of p62, suggesting imbalance in the autophagic process. Active lysosome biogenesis characterizes both LOPD PRE and POST, unparalleled by molecules involved in lysosome tethering (VAMP8, SNAP29, STX17, and GORASP2) and BNIP3. In conclusion this study reveals a specific signature that suggests ERT prolongation and molecular targets to ameliorate patient's outcome.

The glucoamylase from Aspergillus wentii: Purification and characterization.

This study describes for the first time the purification and characterization of a glucoamylase from Aspergillus wentii (strain PG18), a species of the Aspergillus genus Cremei section. Maximum enzyme production (∼3.5 U/ml) was obtained in submerged culture (72 h) with starch as the carbon source, at 25°C, and with orbital agitation (100 rpm). The enzyme was purified with one-step molecular exclusion chromatography. The 86 kDa purified enzyme hydrolyzed starch in a zymogram and had activity against p-nitrophenyl α- d-glucopyranoside. The optimal enzyme pH and temperature were 5.0 and 60°C (at pH 5.0), respectively. The Tm of the purified enzyme was 60°C, at pH 7.0. The purified glucoamylase had a KM for starch of 1.4 mg/ml and a Vmax of 0.057 mg/min of hydrolyzed starch. Molybdenum activated the purified enzyme, and sodium dodecyl sulfate inhibited it. A thin layer chromatography analysis revealed glucose as the enzyme's main starch hydrolysis product. An enzyme's peptide sequence was obtained by mass spectrometry and used to retrieve a glucoamylase within the annotated genome of A. wentii v1.0. An in silico structural model revealed a N-terminal glycosyl hydrolases family 15 (GH15) domain, which is ligated by a linker to a C-terminal carbohydrate-binding module (CBM) from the CBM20 family.

Screening and evaluation of Monascus purpureus FJMR24 for enhancing the raw material utilization rate in rice wine brewing.

BACKGROUND: The rapid development of the rice wine industry has increased the demand for raw materials worldwide. A fungal strain with good adaptability to rice wine brewing conditions, which can also enhance the utilization rate of raw materials (URRM), thus increasing the production efficiency, was sought in the present research. RESULTS: The strain FJMR24 was successfully isolated and screened from 35 fermentation starters and exhibited high amylase activity (2200.9 ± 18.5 U g-1 ) and high glucoamylase activity (2330.4 ± 31.9 U g-1 ). Based on a morphological examination and a sequence analysis of the internal transcribed spacer (ITS) gene and ß-tubulin gene, FJMR24 was identified as Monascus purpureus, which is an edible and versatile fungus that plays a dominant role in the processing of Hong Qu. A moderate pH of 5-6 under incubation at 35 °C for 5-6 days was favorable for the growth and enzyme production of FJMR24. The strain could also tolerate the extreme conditions of 15-45 °C, 18% ethanol (v/v), and an acidity of pH 2. The excellent fermentation adaptability of FJMR24 might enable it to retain high enzyme activity during rice wine brewing. As a result of the action of FJMR24, the URRM of the base liquor increased by around 26% due to increased starch hydrolysis efficiency, which was mainly due to the high unit enzyme activity of FJMR24. CONCLUSION: This study provides perspectives for the application of a M. purpureus strain with high starch hydrolysis activity for enhancing the URRM in traditional rice wine brewing. © 2020 Society of Chemical Industry.

Dietary 5,6,7-Trihydroxy-flavonoid Aglycones and 1-Deoxynojirimycin Synergistically Inhibit the Recombinant Maltase-Glucoamylase Subunit of α-Glucosidase and Lower Postprandial Blood Glucose.

1-Deoxynojirimycin (1-DNJ) is the major effective component of mulberry leaves, exhibiting inhibitory activity against α-glucosidase. However, due to the low content of 1-DNJ in mulberry products, its level cannot meet the lowest dose to exhibit its activity. In this study, a combination of dietary 5,6,7-trihydroxy-flavonoid aglycones with 1-DNJ showed synergistic inhibitory activity against maltase of mice α-glucosidase and recombinant C- and N-termini of maltase-glucoamylase (MGAM) and baicalein with 1-DNJ exhibited the strongest synergistic effect. The synergistic effect of the combination was also confirmed by the maltose tolerance test in vivo. Enzyme kinetics, molecular docking, fluorescence spectrum, and circular dichroism spectrometry studies indicated that the major mechanism of the synergism is that baicalein was a positive allosteric inhibitor and bound to the noncompetitive site of MGAM, causing an increase of the binding affinity of 1-DNJ to MGAM. Our results might provide a theoretical basis for the design of dietary supplements containing mulberry products.

Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.

OBJECTIVE: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass. RESULTS: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0-9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT. CONCLUSIONS: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.

Spectrum of amyloglucosidase mutations in Asian Indian patients with Glycogen storage disease type III.

Glycogen storage disease type III (GSD III) is a rare autosomal recessive inborn error of glycogen degradation pathway due to deficiency or reduced activity of glycogen debranching enzyme (GDE) that results in accumulation of abnormal glycogen in the liver, muscle, and heart. The cardinal hallmarks are hepatomegaly, fasting hypoglycemia, seizures, growth retardation, progressive skeletal myopathy, and cardiomyopathy in few. To date, 258 mutations in amyloglucosidase (AGL) gene have been identified worldwide. However, the mutation spectrum in the Asian Indian region is yet to be well characterized. We investigated 24 patients of Asian origin from 21 unrelated families with a provisional diagnosis of GSD III based on clinical and biochemical criteria. Molecular diagnosis was assessed by bidirectional sequencing and the impact of novel missense variants on the tertiary (three-dimensional) structure of GDE was evaluated by molecular modeling approach. Eighteen different pathogenic variants were identified, out of which 78% were novel. Novel variants included five nonsense, three small duplications and two small deletions, a splice site variant, and three missense variants. Variations in Exons 4, 14, 19, 24, 27, and 33 accounted for 61% of the total pathogenic variants identified and Allele p.Gly798Alafs*3 showed a high allele frequency of 11%. Molecular modeling study of novel pathogenic missense variants indicated the probable underlying molecular mechanism of adverse impact of variations on the structure and catalytic function of human GDE. Our study is the first large study on GSD III from the Asian subcontinent, which further expands the mutation spectrum of AGL.

Zearalenone Removal from Corn Oil by an Enzymatic Strategy.

The estrogen-like mycotoxin zearalenone (ZEN) is one of the most widely distributed contaminants especially in maize and its commodities, such as corn oil. ZEN degrading enzymes possess the potential for counteracting the negative effect of ZEN and its associated high safety risk in corn oil. Herein, we targeted enhancing the secretion of ZEN degrading enzyme by Pichia pastoris through constructing an expression plasmid containing three optimized expression cassettes of zlhy-6 codon and signal peptides. Further, we explored various parameters of enzymatic detoxification in neutralized oil and analyzed tocopherols and sterols losses in the corn oil. In addition, the distribution of degraded products was demonstrated as well by Agilent 6510 Quadrupole Time-of-Flight mass spectrometry. P. pastoris GSZ with the glucoamylase signal was observed with the highest ZLHY-6 secretion yield of 0.39 mg/mL. During the refining of corn oil, ZEN in the crude oil was reduced from 1257.3 to 13 µg/kg (3.69% residual) after neutralization and enzymatic detoxification. Compared with the neutralized oil, no significant difference in the total tocopherols and sterols contents was detected after enzymatic detoxification. Finally, the degraded products were found to be entirely eliminated by washing. This study presents an enzymatic strategy for efficient and safe ZEN removal with relatively low nutrient loss, which provides an important basis for further application of enzymatic ZEN elimination in the industrial process of corn oil production.

Novel Mutations Found in Individuals with Adult-Onset Pompe Disease.

Pompe disease, or glycogen storage disease II is a rare, progressive disease leading to skeletal muscle weakness due to deficiency of the acid α-1,4-glucosidase enzyme (GAA). The severity of disease and observed time of onset is subject to the various combinations of heterozygous GAA alleles. Here we have characterized two novel mutations: c.2074C>T and c.1910_1918del, and a previously reported c.1082C>G mutation of uncertain clinical significance. These mutations were found in three unrelated patients with adult-onset Pompe disease carrying the common c.-32-13T>G mutation. The c.2074 C>T nonsense mutation has obvious consequences on GAA expression but the c.1910_1918del (deletion of 3 amino acids) and c.1082C>G missense variants are more subtle DNA changes with catastrophic consequences on GAA activity. Molecular and clinical analyses from the three patients corresponded with the anticipated pathogenicity of each mutation.

Spatial induction of genes encoding secreted proteins in micro-colonies of Aspergillus niger.

Aspergillus niger is used by the industry to produce enzymes and metabolites such as citric acid. In liquid cultures, it can grow as a dispersed mycelium or as micro-colonies with a width in the micrometer to millimeter range. Here, it was assessed whether expression of genes encoding secreted enzymes depends on mycelium morphology. To this end, expression of the reporter gene gfp from the promoters of the glucoamylase gene glaA, the feruloyl esterase gene faeA and the α-glucuronidase gene aguA was causally related to micro-colony size within a liquid shaken culture. Data could be fitted by hyperbolic functions, implying that the genes encoding these secreted proteins are expressed in a shell at the periphery of the micro-colony. The presence of such a shell was confirmed by confocal microscopy. Modelling predicted that the width of these zones was 13 to 156 µm depending on growth medium and micro-colony diameter. Together, data indicate that the highest productive micro-colonies are those colonies that have a radius ≤ the width of the peripheral expression zone.

Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris.

Brown-rot fungi preferentially degrade softwood and cause severe breakdown of wooden structures. At the initial stage of the brown-rot decay, penetrating hyphae of the fungi are observed in ray parenchyma. Since starch grains are known to be present in the ray parenchyma of sapwood, investigation of the functions and roles of the starch-degrading enzymes is important to understand the initial stage of brown-rot decay. We purified and characterized two starch-degrading enzymes, an α-amylase (FpAmy13A) and a glucoamylase (FpGLA15A), from the brown-rot fungus, Fomitopsis palustris, and cloned the corresponding genes. The optimal temperature for both enzymes was 60 °C. FpAmy13A showed higher activity at a broad range of pH from 2.0 to 5.0, whereas FpGLA15A was most active at pH 5.0-6.0. Notable thermal stability was found for FpGLA15A. Approximately 25% of the activity remained even after treatment at 100 °C for 30 min in sodium phosphate buffer at pH 7.0. These different characteristics imply the different roles of these enzymes in the starch degradation of wood.