Xiangru Liao

Decolorization of synthetic dyes by crude laccase from a newly isolated Trametes trogii strain cultivated on solid agro-industrial residue

A new dye-decolorizing white-rot fungus was isolated and identified as Trametes trogii based on its ITS-5.8S rRNA gene sequence analysis and morphological characteristics. Laccase was the only lignolytic enzyme produced by this strain during solid substrate fermentation (SSF) in soybean cake, a solid agro-industrial residue used for the first time in enzyme production. The extracellular crude enzyme from T. trogii in solid substrate fermentation showed good activity in synthetic dye color removal, decolorizing 85.2% Remazol Brilliant Blue R (50 mg l(-1)), 69.6% Reactive Blue 4 (35 mg l(-1)), and 45.6% Acid Blue 129 (83.3 mg l(-1)) without the addition of redox mediators, 90.2% Acid Red 1 (10 mg l(-1)), and 65.4% Reactive Black 5 (18.3 mg l(-1)) with the addition of 1mM 1-hydroxybenzotriazole in 30 min. Native polyacrylamide gel electrophoresis (Native-PAGE) of the crude enzyme and effects of laccase inhibitors on decolorization corroborated the laccase as the major enzyme involved in the decolorization of dyes. The comparison of color removal by the crude culture filtrates and by the whole fungal culture on the solid substrate revealed the former was more advantageous.

Optimizing the codon usage of synthetic gene with QPSO algorithm

Molecular Biology makes it possible to express foreign genes in microorganism, plants and animals. To improve the heterologous expression, it is important that the codon usage of sequence be optimized to make it adaptive to host organism. In this paper, a novel method based on Quantum-behaved Particle Swarm Optimization (QPSO) algorithm is developed to optimize the codon usage of synthetic gene. Compared to the existing probability methods, QPSO is able to generate better results when DNA/RNA sequence length is less than 6Kb which is the commonly used range. While the software or web service based on probability method may not exclude all defined restriction sites when there are many undesired sites in the sequence, our proposed method can remove the undesired site efficiently during the optimization process.

FUNGAL PRETREATMENT OF SWITCHGRASS FOR IMPROVED SACCHARIFICATION AND SIMULTANEOUS ENZYME PRODUCTION

This study investigates fungal pretreatment of switchgrass involving solid state fermentation (SSF) to improve saccharification and simultaneously produce enzymes as co-products. The results revealed that the fungus Pycnoporus sp. SYBC-L3 can significantly degrade lignin and enhance enzymatic hydrolysis efficiency. After a 36-d cultivation period, nearly 30% reduction in lignin content was obtained without significant loss of cellulose and hemicellulose, while a considerable amount of laccase, as high as 6.3 U/g, was produced. After pretreatment, pores on switchgrass surface were observed using scanning electron microscopy (SEM). The enzymatic hydrolysis efficiency for the switchgrass with 36-d pretreatment was about 50% greater than the untreated one. Our results suggest that solid state fungal cultivation may be a good method for switchgrass pretreatment, which can simultaneously achieve high efficiency of enzymatic hydrolysis and production of some useful enzymes for other industrial utilization.

Isolation and identification of a new hypocrellin A-producing strain Shiraia sp. SUPER-H168

A new hypocrellin A-producing strain, Shiraia sp. SUPER-H168, was isolated from tissues of bamboo, Brachystachyum densiflorum. The morphology of this strain was characterized with a light microscope and a scanning electronic microscope. The mycelia, conidia, pycnidia of fungus were observed. Small pycnidia (10-20 microm in length) full of small conidia appeared on the mycelia, which were first reported in this study. The 18S rDNA region of this strain was amplified and sequenced. Then a neighbor-joining tree of 18S rDNA was constructed. According to the result of analysis, the strain SUPER-H168 was proved to belong to the genus Shiraia. Hypocrellin A was produced by solid-state fermentation, extracted by acetone, isolated by preparative RP-HPLC, and identified by RP-HPLC, ESI-MS and ultraviolet-visible absorbing scanning with diode array detection. The HA production could reach 2.02 mg/g dry solid substrate.

Induction of hypocrellin production by Triton X-100 under submerged fermentation with Shiraia sp. SUPER-H168.

Hypocrellins are important photodynamic therapy compounds for cancer disease. The effect of surfactants on hypocrellin production of Shiraia sp. SUPER-H168 was evaluated under submerged fermentation condition. The production of hypocrellins could reach 780.6 mg/l with the addition of Triton X-100, confirmed by color reaction, high performance liquid chromatography, electrospray ionization mass spectrometry and nuclear magnetic resonance experiments. According to our observation, treatment of the culture at the beginning of the fermentation was most effective, and the yield of hypocrellins was much lower with the addition of Triton X-100 during the log phase and stationary phase. Shiraia sp. SUPER-H168 could not produce hypocrellin with the addition of other tested surfactants, such as Tween 40, Triton X-114 and SDS. The experimental results indicated that Shiraia sp. SUPER-H168 could not produce hypocrellins without Triton X-100 under submerged fermentation condition.

Production, characterization, cloning and sequence analysis of a monofunctional catalase from Serratia marcescens SYBC08

A monofunctional catalase from Serratia marcescens SYBC08 produced by liquid state fermentation in 7 liter fermenter was isolated and purified by ammonium sulfate precipitation (ASP), ion exchange chromatography (IEC), and gel filtration (GF) and characterized. Its sequence was analyzed by LC‐MS/MS technique and gene cloning. The highest catalase production (20,289 U · ml–1) was achieved after incubation for 40 h. The purified catalase had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the catalase (199,584 U · mg–1 protein) was 3.44 times higher than that of Halomonas sp. Sk1 catalase (57,900 U · mg–1 protein). The enzyme without peroxidase activity was found to be an atypical electronic spectrum of monofunctional catalase. The apparent Km and Vmax were 78 mM and 188, 212 per µM H2O2 µM heme–1 s–1, respectivly. The enzyme displayed a broad pH activity range (pH 5.0–11.0), with optimal pH range of 7.0–9.0: It was most active at 20 °C and had 78% activity at 0 °C. Its thermo stability was slightly higher compared to that of commercial catalase from bovine liver. LC–MS/MS analysis confirmed that the deduced amino acid sequence of cloning gene was the catalase sequence from Serratia marcescens SYBC08. The sequence was compared with that of 23 related catalases. Although most of active site residues, NADPH‐binding residues, proximal residues of the heme, distal residues of the heme and residues interacting with a water molecule in the enzyme were well conserved in 23 related catalases, weakly conserved residues were found. Its sequence was closely related with that of catalases from pathogenic bacterium in the family Enterobacteriaceae. This result imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage. Calalase yield by Serratia marcescens SYBC08 has potential industrial application in scavenging hydrogen peroxide. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Antifungal Activity of Isolated Bacillus amyloliquefaciens SYBC H47 for the Biocontrol of Peach Gummosis.

The gummosis disease is caused by Botryosphaeria dothidea (Moug. ex. Fr) Ces. et de Not., and it is one of the most important diseases of stone fruits worldwide. The use of biocontrol as an alternative approach to synthetic chemical fungicides has aroused general concern about how to control plant diseases that are caused by phytopathogens. The aim of this study is to isolate Bacillus strains from raw honeys with the capacity to inhibit B. dothidea and to explore the mechanisms by which they could be used in the biocontrol of peach gummosis. Bacillus amyloliquefaciens SYBC H47 was isolated and identified on the basis of its physiological and biochemical characteristics and its 16S rRNA and gyrB gene sequences. The cell suspension and the cell-free supernatant of its culture showed significant antifungal activity against Aspergillus niger, Mucor racemosus, Fusarium oxysporum, Penicillium citrinum, and Candida albicans by agar-diffusion assays. The primary antifungal substances were bacillomycin L, fengycin, and surfactin, which were analyzed by HPLC LC/ESI-MS/MS. Bacillomycin L showed the best inhibitory effect against conidial germination of B. dothidea, followed by fengycin and surfactin. Surfactin had limited effects on mycelial growth, contrary to those of bacillomycin L and fengycin. However, a mixture of the three lipopeptides had a synergistic effect that disrupted the structure of the conidia and mycelia. In order to reduce the production cost, the use of waste frying peanut oil and soy oil as the sole carbon source increased the lipopeptide yield levels by approximately 17% (2.42 g/L) and 110% (4.35 g/L), respectively. In a field trial, the decreases in the infected gummosis rate (IGR) and the disease severity index (DSI) through cell suspension treatments were 20% and 57.5% (in 2014), respectively, and 40% and 57.5% (in 2015), respectively, in comparison with the control. In conclusion, B. amyloliquefaciens SYBC H47 could inhibit the germination of conidia and the growth of mycelia from B. dothidea; therefore, this strain behaves as a potential biocontrol agent against the gummosis disease.

Serratia marcescens SYBC08 catalase isolated from sludge containing hydrogen peroxide shows increased catalase production by regulation of carbon metabolism

A high‐catalase‐producing strain, which was isolated from sludge containing hydrogen peroxide, was identified as Serratia marcescens SYBC08 by 16S rDNA sequence analysis. Serratia spp. was reported as non‐spore‐forming bacterium (except S. marcescens spp. sakuensis), but in our study electron microscopic observation revealed that the strain did produce spores. The content of the main fatty acid C16:0 (14.8%) was significantly different from that of S. marcescens spp. sakuensis (33.2%) and S. marcescens spp. marcescens DSM 30121T (34.8%), and the biochemical characteristics were not identical to those of S. marcescens spp. sakuensis. We speculate that the relatively high catalase activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. The most suitable carbon and nitrogen sources for the catalase production by S. marcescens SYBC08 were citric acid and corn steep liquor powder. A strategy of carbon metabolism regulation to enhance the catalase production was exploited. In the 7‐L fermenter, catalase production (20 353 U/mL) obtained in the presence of glucose and citric acid was 1.68‐ and 1.31‐fold higher than that obtained in the presence of glucose or citric acid, at equimolar carbon concentration. This production yield was much higher than that of many catalase‐producing strains, but only slightly lower than the production by Micrococcus luteus (34 601 U/mL). The results suggest that the new spore‐forming S. marcescens SYBC08 is a potential candidate for the production of catalase.

Adaptive Responses to Oxidative Stress in the Filamentous Fungal Shiraia bambusicola

Shiraia bambusicola can retain excellent physiological activity when challenged with maximal photo-activated hypocrellin, which causes cellular oxidative stress. The protective mechanism of this fungus against oxidative stress has not yet been reported. We evaluated the biomass and hypocrellin biosynthesis of Shiraia sp. SUPER-H168 when treated with high concentrations of H2O2. Hypocrellin production was improved by nearly 27% and 25% after 72 h incubation with 10 mM and 20 mM H2O2, respectively, while the inhibition ratios of exogenous 20 mM H2O2 on wild S. bambusicola and a hypocrellin-deficient strain were 20% and 33%, respectively. Under exogenous oxidative stress, the specific activities of catalase, glutathione reductase, and superoxide dismutase were significantly increased. These changes may allow Shiraia to maintain normal life activities under oxidative stress. Moreover, sufficient glutathione peroxidase was produced in the SUPER-H168 and hypocrellin-deficient strains, to further ensure that S. bambusicola has excellent protective abilities against oxidative stress. This study creates the possibility that the addition of high H2O2 concentrations can stimulate fungal secondary metabolism, and will lead to a comprehensive and coherent understanding of mechanisms against oxidative stresses from high hydrogen peroxide concentrations in the filamentous fungal Shiraia sp. SUPER-H168.

CRISPR system in filamentous fungi: Current achievements and future directions

As eukaryotes, filamentous fungi share many features with humans, and they produce numerous active metabolites, some of which are toxic. Traditional genetic approaches are generally inefficient, but the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system that has been widely used for basic research on bacteria, mammals and plants offers a simple, fast, versatile technology for systemic research on filamentous fungi. In this review, we summarized the current knowledge on Cas9 and its variants, various selective markers used to screen positive clones, different ways used to detect off-target mutations, and different approaches used to express and transform the CRISPR complex. We also highlight several methods that improve the nuclease specificity and efficiency, and discuss current and potential applications of CRISPR/Cas9 system in filamentous fungi for pathogenesis decoding, confirmation of the gene and pathway, bioenergy process, drug discovery, and chromatin dynamics. We also describe how the synthetic gene circuit of CRISPR/Cas9 systems has been used in the response to various complex environmental signals to redirect metabolite flux and ensure continuous metabolite biosynthesis.