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Background Application of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production. Results A total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C. Conclusions Overall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.

Background Raw starch-degrading enzyme (RSDE) is applied in biorefining of starch to produce biofuels efficiently and economically. At present, RSDE is obtained via secretion by filamentous fungi such as Penicillium oxalicum . However, high production cost is a barrier to large-scale industrial application. Genetic engineering is a potentially efficient approach for improving production of RSDE. In this study, we combined genetic engineering and random mutagenesis of P. oxalicum to enhance RSDE production. Results A total of 3619 mutated P. oxalicum colonies were isolated after six rounds of ethyl methanesulfonate and Co 60 -γ-ray mutagenesis with the strain A2-13 as the parent strain. Mutant TE4-10 achieved the highest RSDE production of 218.6 ± 3.8 U/mL with raw cassava flour as substrate, a 23.2% compared with A2-13. Simultaneous deletion of transcription repressor gene PoxCxrC and overexpression of activator gene PoxAmyR in TE4-10 resulted in engineered strain GXUR001 with an RSDE yield of 252.6 U/mL, an increase of 15.6% relative to TE4-10. Comparative transcriptomics and real-time quantitative reverse transcription PCR revealed that transcriptional levels of major amylase genes, including raw starch-degrading glucoamylase gene PoxGA15A , were markedly increased in GXUR001. The hydrolysis efficiency of raw flour from cassava and corn by crude RSDE of GXUR001 reached 93.0% and 100%, respectively, after 120 h and 84 h with loading of 150 g/L of corresponding substrate. Conclusions Combining genetic engineering and random mutagenesis efficiently enhanced production of RSDE by P. oxalicum . The RSDE-hyperproducing mutant GXUR001 was generated, and its crude RSDE could efficiently degrade raw starch. This strain has great potential for enzyme preparation and further genetic engineering.

Few studies have been conducted on Mycena species in Hunan Province, China. During our research on the species diversity of Mycena in Hunan Province, we identified approximately 30 Mycena species based on morphological and molecular evidence. Five species are recognized herein as new to science, namely, M. fulvomarginata , M. huangsangensis , M. hongfengensis , M. subroriduliformis , and M. roseolamellata . The phylogenetic analyses of a combined ITS and LSU sequence dataset revealed that five new species each formed an independent lineage that could separate phenotypically similar and phylogenetically related species. Descriptions, photographs, and phylogenetic analysis results are provided for the five new species, along with the comparisons with related species. A key to all Mycena species found in Hunan is also provided.

Abstract Phylogenetic relationships between Tuber pseudoexcavatum and other Tuber species were investigated by studying the sequences of four genes: 5.8S-ITS2, β-tubulin, protein kinase C and elongation factor 1α. The four phylogenetic trees allowed to differentiate the black truffle clade, composed of two subclades, one comprising the Asian black truffles (T. indicum, T. sinense, T. himalayense) and the Perigord black truffle (T. melanosporum), the second comprising T. pseudoexcavatum and T. brumale. These two subclades diverged relatively early. We propose a common ancestor, located between Europe and China, to all the black truffles. The T. brumale/pseudoexcavatum subclade would have started to diverge and migrate first, T. brumale towards Europe through a northern route and T. pseudoexcavatum towards China. Later the T. melanosporum subclade would have started to migrate through the same route, T. melanosporum towards Europe and T. indicum towards China, leading to vicariant species.

Raw starch-degrading enzyme (RSDE) is applied in biorefining of starch to produce biofuels efficiently and economically. At present, RSDE is obtained via secretion by filamentous fungi such as Penicillium oxalicum. However, high production cost is a barrier to large-scale industrial application. Genetic engineering is a potentially efficient approach for improving production of RSDE. In this study, we combined genetic engineering and random mutagenesis of P. oxalicum to enhance RSDE production. A total of 3619 mutated P. oxalicum colonies were isolated after six rounds of ethyl methanesulfonate and Co.sup.60-[gamma]-ray mutagenesis with the strain A2-13 as the parent strain. Mutant TE4-10 achieved the highest RSDE production of 218.6 [+ or -] 3.8 U/mL with raw cassava flour as substrate, a 23.2% compared with A2-13. Simultaneous deletion of transcription repressor gene PoxCxrC and overexpression of activator gene PoxAmyR in TE4-10 resulted in engineered strain GXUR001 with an RSDE yield of 252.6 U/mL, an increase of 15.6% relative to TE4-10. Comparative transcriptomics and real-time quantitative reverse transcription PCR revealed that transcriptional levels of major amylase genes, including raw starch-degrading glucoamylase gene PoxGA15A, were markedly increased in GXUR001. The hydrolysis efficiency of raw flour from cassava and corn by crude RSDE of GXUR001 reached 93.0% and 100%, respectively, after 120 h and 84 h with loading of 150 g/L of corresponding substrate. Combining genetic engineering and random mutagenesis efficiently enhanced production of RSDE by P. oxalicum. The RSDE-hyperproducing mutant GXUR001 was generated, and its crude RSDE could efficiently degrade raw starch. This strain has great potential for enzyme preparation and further genetic engineering.

Mycena (Pers.) Roussel (1806) is a large genus of Mycenaceae known for having small to medium-sized basidiomata. It is typified by the species Mycena galericulata (Scop.) Gray. For years, many mycologists have made important contributions to understanding Mycena and several monographs have been published. Three specimens were collected from China that belonged to the genus Mycena . On the basis of morphological analysis and phylogenetic analyses employing DNA sequences, a new species is described. Mycena brunnescens sp. nov. is described as a new species from subtropical areas of China. It is characterised by its brown pileus, whitish lamellae that turns brown when bruised, orange to brown lamellae edges, the absence of pleurocystidia and cheilocystidia with simple or branched excrescences at the apex containing yellowish-brown contents. We performed phylogenetic analyses on a concatenated dataset comprising the internal transcribed spacer and large subunit regions of nuclear ribosomal RNA using Bayesian Inference and Maximum Likelihood methods. The result showed that the new taxon clustered in an independent group and is closely related to M. albiceps and M. flosoides .

(Pers.) Roussel (1806) is a large genus of Mycenaceae known for having small to medium-sized basidiomata. It is typified by the species (Scop.) Gray. For years, many mycologists have made important contributions to understanding and several monographs have been published. Three specimens were collected from China that belonged to the genus . On the basis of morphological analysis and phylogenetic analyses employing DNA sequences, a new species is described. sp. nov. is described as a new species from subtropical areas of China. It is characterised by its brown pileus, whitish lamellae that turns brown when bruised, orange to brown lamellae edges, the absence of pleurocystidia and cheilocystidia with simple or branched excrescences at the apex containing yellowish-brown contents. We performed phylogenetic analyses on a concatenated dataset comprising the internal transcribed spacer and large subunit regions of nuclear ribosomal RNA using Bayesian Inference and Maximum Likelihood methods. The result showed that the new taxon clustered in an independent group and is closely related to and .

Lead（Pb）isotopes have been extensively employed in tracing sources of Pb and its transport pathways through the environment.However,Pb isotopic ratios in related geochemical reference materials are scarce.Here,we report high-precision Pb isotopic ratios measured by Nu Plasma II MC-ICP-MS using calibrated ^205）Tl/^203Tl=2.38865（NIST SRM 997）for mass discrimination correction.The long-term external precision（2SD）for NISTSRM 981 of Pb,BCR-2,and BHVO-2 are 0.31‰（n=105）,0.42‰（n=11）,and 0.25‰（n=5）for ^208Pb/^206Pb and 0.16‰,0.53‰,and 0.07‰for ^206Pb/^207Pb,both respectively,and their Pb isotopic ratios are in excellent agreement with the recommended values.Using this method,we report for the first time Pb isotopic compositions in shale SGR-1b（USGS）;coal CLB-1（USGS）;stream sediments GSD-17,-21,and-23（IGGE）;soils GSS-12,-13,-14,-15,and-16（IGGE）;plants GSV-1,-2,and-3（IGGE）;and human hair GSH-1（IGGE）.

To the Editor： We reported 2 cases ofectopic thyroid gland （ETG） masquerading as pituitary adenoma （PA）. The first case was a 15-year-old boy with growth, and mental retardation whose height was 121 cm and weight was 44 kg on admission. Bone age development index was 40, while bone age showed 6-10 years old.

Intracloud (IC) lightning flashes have been simulated in fine resolution (12.5 m) by using a bidirectional stochastic lightning parameterization scheme within 2-dimensional domain. The simulated results show that the IC flashes have a bilevel channel structure and the altitudes of the horizontal channels are at the same heights of potential wells, which are supported by the previous VHF source observations and balloon soundings of electric field profile in the thundercloud. Further conclusions are: (1) After an IC flash is initiated near the boundary between positve and nagetive charge zone, the negative (or positive) leader tends to propagate into the positive (or negative) charge zone.Both types of positive and negative IC flashes have been reproduced and their polarity depends on the up and down disposition of the positive and negative charge regions. (2) The extension range of leaders is correlative with the cloud charge distribution. The leader is possible to extend through the inverted charge region all over where it is extending, but keeps away from the isolated charge area of the same polarity. (3) The channel structures also depend on the electric potential distributions in the thundercloud. Before propagating into the central area of potential wells, the leader tends to extend along the direction with the maximum of potential gradient. Once extending away from the center of potential wells, the leader tends to extend along the direction with the slowest potential change. (4)The IC flash channels have the fractal feature with fractal dimension 1.45 before leaders pass through the central area of charge regions. The exponent decreases rapidly once leaders extend into the low-density charge regions. (5) The induced charges of opposite polarity are deposited in the leader channels within preexisting positive and negative charge regions during IC flash discharges. This causes a new and complicated charge distribution in the thundercloud, and the potential extremum drops from 200 to 20 Mv when the IC flash terminates.