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

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*

Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.
Codon/genetics* ; Humans ; Pichia/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales ; Vascular Endothelial Growth Factor A/genetics* ; Vascular Endothelial Growth Factors

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

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

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

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (~28%) and well above the predicted error of Sanger sequencing (~2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.
DNA* ; Gene Deletion* ; Mutation Rate ; Open Reading Frames ; Polymerase Chain Reaction ; Saccharomycetales ; Schizosaccharomyces*

Cryptococcosis is caused by several Cryptococcus species, including C. neoformans and C. gattii. Skin involvement is seen in 10~20% of systemic cryptococcosis. There are also rare cases of primary cutaneous cryptococcosis in which skin-penetrating trauma is the alleged mechanism of infection. A 16-year-old male presented with multiple, 0.2~0.3 cm-sized, brownish papules on the whole body for 2 weeks. He had past history of acute lymphoblastic leukemia and received bone marrow transplant 1 year ago. After leukemia had recurred 1 month ago and after chemotherapy, multiple brownish papules developed. Histopathologic examinations revealed narrow-based budding yeasts in hematoxylin and eosin, Periodic acid-Schiff, and Gomori methenamine silver stains. Also in mucicarmine stain there were pink-colored capsules around the cell walls. Finally it was diagnosed as deep fungal infection due to Cryptococcus species. In spite of administrating fluconazole, the patient expired due to respiratory failure caused by pneumonia. Herein, we report a case of Cryptococcus species infection in a bone marrow transplant patient.
Adolescent ; Bone Marrow* ; Capsules ; Cell Wall ; Coloring Agents ; Cryptococcosis ; Cryptococcus* ; Drug Therapy ; Eosine Yellowish-(YS) ; Fluconazole ; Hematoxylin ; Humans ; Leukemia ; Male ; Methenamine ; Pneumonia ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Respiratory Insufficiency ; Saccharomycetales ; Skin

No abstract available.
Candidemia* ; Saccharomycetales*