No abstract available.
Candidemia* ; Saccharomycetales*

Nowadays, engineered Komagataella phaffii plays an important role in the biosynthesis of small molecule metabolites and protein products, showing great potential and value in industrial productions. With the development and application of new editing tools such as CRISPR/Cas9, it has become possible to engineer K. phaffii into a cell factory with high polygenic efficiency. Here, the genetic manipulation techniques and objectives for engineering K. phaffii are first summarized. Secondly, the applications of engineered K. phaffii as a cell factory are introduced. Meanwhile, the advantages as well as disadvantages of using engineered K. phaffii as a cell factory are discussed and future engineering directions are prospected. This review aims to provide a reference for further engineering K. phaffii cell factory, which is supposed to facilitate its application in bioindustry.
Saccharomycetales/genetics* ; Genetic Techniques

Scleroglucan is a high-molecular water-soluble microbial exopolysaccharide and mainly applied in the fields of petroleum, food, medicine and cosmetics. The high molecular weight of scleroglucan produced by microbial fermentation leads to low solubility, high viscosity and poor dispersibility, thus bringing a series of difficulties to extraction, preservation and application. It is important to explore suitable degradation method to adjust the molecular weight of scleroglucan for expanding its industrial application. Taking Sclerotium rolfsii WSH-G01 as a model strain, in which functional annotations of the glucanase genes were conducted by whole genome sequencing. Based on design of culture system for culture system for differential expression of β-glucanase, endogenous β-glucanase genes in S. rolfsii WSH-G01 were excavated by transcriptomics analysis. Functions of these potential hydrolases were further verified. Finally, 14 potential endogenous hydrolase genes were obtained from S. rolfsii. After heterologous overexpression in Pichia pastoris, 10 soluble enzymes were obtained and 5 of them had the activity of laminarin hydrolysis by SDS-PAGE and enzyme activity analysis. Further investigation of the 5 endogenous hydrolases on scleroglucan degradation showed that enzyme GME9860 has positive hydrolysis effect. The obtained results provide references not only for obtaining low and medium molecular weight of scleroglucan with enzymatic hydrolysis, but also for producing different molecular weight of scleroglucan during S. rolfsii fermentation process with metabolic engineering.
Basidiomycota/genetics* ; Glucans ; Hydrolysis ; Saccharomycetales

Thirty-nine patients with pityrosporum folliculitis were investigated clinically and histopathologically. On clinical observation there were numerous, chronic, moderately itchy (64.1%), dome-shaped papules (89.7%) and pustules (66.7%). The most frequent sites of the lesions were the upper portion of the chest (76.9%) and back (56.4%). In biopsy specimens, abundant round and budding yeast cells were seen in a dilated hair follicle. The reptured follicle was observed in 19 specimen (48.7%). The accumulation on inflammatory cells were observed in or around the upper part of the follicle in all specimens. The effect of antimycotic treatment was excellent. After 4 weeks of treatment, 36 patients (92%) were cured and 3 (8%) had improved significantly. KOH/Parker Ink direct smear was done in 20 patients. Blue-colored round and budding yeast cells were observed under a light mcroscope in all patients. We suggest that pityrosporum folliculitis is a common disease of young and middle-aged Koreans.
Biopsy ; Folliculitis* ; Hair Follicle ; Humans ; Ink ; Malassezia* ; Saccharomycetales ; Thorax ; Yeasts

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.
6-Phytase/genetics* ; Pichia/genetics* ; Plasmids ; Recombinant Proteins/genetics* ; Saccharomycetales

Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.
Humans ; Kinetics ; Mutation ; Phospholipases A2, Secretory ; Phospholipids ; Saccharomycetales ; Substrate Specificity

Fatty acids (FA) are widely used as feed stocks for the production of cosmetics, personal hygiene products, lubricants and biofuels. Ogataea polymorpha is considered as an ideal chassis for bio-manufacturing, due to its outstanding characteristics such as methylotroph, thermal-tolerance and wide substrate spectrum. In this study, we harnessed O. polymorpha for overproduction of fatty acids by engineering its fatty acid metabolism and optimizing the fermentation process. The engineered strain produced 1.86 g/L FAs under the optimized shake-flask conditions (37℃, pH 6.4, a C/N ratio of 120 and an OD₆₀₀ of seed culture of 6-8). The fed-batch fermentation process was further optimized by using a dissolved oxygen (DO) control strategy. The C/N ratio of initial medium was 17.5, and the glucose medium with a C/N ratio of 120 was fed when the DO was higher than 30%. This operation resulted in a titer of 18.0 g/L FA, indicating the potential of using O. polymorpha as an efficient cell factory for the production of FA.
Culture Media ; Fatty Acids ; Fermentation ; Metabolic Engineering ; Saccharomycetales/metabolism*

Primary cutaneous cryptococcosis (PCC) is a localized skin infection confined to one body region, without evidence of dissemination. The clinical presentation of PCC is so variable that its diagnosis requires a high index of suspicion. A 52-year-old woman presented with localized grouped erythematous papulovesicles on the left ear lobe for 6 months with wax and wane pattern. However, there were no signs of systemic cryptococcal infection. Histopathological examination showed numerous encapsulated round spores and budding yeasts in the dermis. Culture of aspirate from the wound and tissue samples revealed Cryptococcus neoformans. Herein, we report an interesting case of PCC on the left ear that clinically mimics herpes zoster.
Body Regions ; Cryptococcosis ; Cryptococcus neoformans ; Dermis ; Ear ; Female ; Herpes Zoster ; Humans ; Saccharomycetales ; Skin ; Spores

We report a case of primary cutaneous cryptococcosis in a 63-year-old female, who had a painful deep ulceration on the left forearm for 2 months. Histopathological examination showed numerous encapsulated round spores in the dermis and subcutis. A tissue culture on Sabouraud's media without cycloheximide showed whitish creamy colonies and revealed budding yeast cells in lactophenol cotton blue preparations. The organism was identified as Cryptococcus neoformans. The lesion was successfully treated with oral fluconazole(400 mg/day) for 2 months combined with surgical debridement.
Cryptococcosis* ; Cryptococcus neoformans ; Cycloheximide ; Debridement ; Dermis ; Female ; Fluconazole* ; Forearm ; Humans ; Middle Aged ; Saccharomycetales ; Spores ; Ulcer

The clinical picture and CSF findings in cryptococcal meningitis may be identical with those of tuberculous meningitis. The differential diagnosis can be made by finding the budding yeast organism in the counting chamber of in stained smear, the detection of cryptococcal antigen in CSF by the latex agglutination test, and by culture of the fungus on Sabouraud agar. We experienced a case of cryptococcal meningitis in the 48 years old woman, which was confirmed by Indian ink preparation and culture.
Agar ; Diagnosis, Differential ; Female ; Fungi ; Humans ; Ink ; Latex Fixation Tests ; Meningitis, Cryptococcal* ; Saccharomycetales ; Tuberculosis, Meningeal