Optimizing Pichia pastoris Protein Secretion: Role of N-Linked Glycosylation on the α-Mating Factor Secretion Signal Leader.

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.

Effects of hepatocyte growth factor-transfected mesenchymal stem cell transplantation in canine injured vocal folds.

This study aimed to assess the efficacy of hepatocyte growth factor (HGF)-transfected adipose-derived mesenchymal stem cell (ADSC) transplantation in the injured vocal folds (VFs) of canines. A lentiviral vector encoding HGF was successfully produced via Gateway cloning, which was used to infect ADSCs. Four weeks after transoral laser microsurgery (type II) with CO2 laser, the beagles of each group were injected with HGF-transfected ADSCs or uninfected ADSCs into VFs. The results showed that the retention of HGF-transfected ADSCs in the VFs persisted about three months post-injection. The VFs in the HGF-transfected ADSCs group exhibited a closer-to-normal structure with less collagen deposition and higher amounts of hyaluronic acid (HA) in the third month. The short microvilli in the HGF-transfected ADSCs group showed a dense and uniform distribution. These results revealed that HGF-transfected ADSC is a potential treatment option for injured VFs.

Surgical and Functional Outcome After Endoscopic Resection Via Transthyrohyoid for Early Glottic Cancer.

Transthyrohyoid access to the larynx for endoscopic resection (TTER) for early-stage glottic cancer in patients with difficult laryngeal exposure (DLE) has recently been developed. However, little is known about the postoperative conditions of patients. Twelve early-stage glottic cancer patients with DLE who received TTER were retrospectively reviewed. Clinical information was collected during the perioperative period. Functional outcome was evaluated using Voice Handicap Index-10 (VHI-10) and Eating Assessment Tool-10 (EAT-10) preoperatively and 12 months after surgery. None of the patients experienced serious complications after TTER. The tracheotomy tube was removed in all patients. The 3-year local control rate was 91.6%. The VHI-10 score decreased from 18.92 to 11.75 (p < .001), and the EAT-10 scores of the 3 patients changed slightly. Thus, TTER may be a good option for early-stage glottic cancer patients with DLE.

The α-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii.

BACKGROUND: The budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii. RESULTS: In this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression. CONCLUSIONS: The screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.

Isolation and evaluation of strong endogenous promoters for the heterologous expression of proteins in Pichia pastoris.

BACKGROUND: The heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production usually requires to express more than one gene in the host cells. In eukaryotes, the pathway flux is typically balanced by controlling the transcript levels of the genes involved. It is difficult to balance the stoichiometric fine-tuning of the reaction steps of the pathway by acting on one or two promoters. Furthermore, the promoter used should not be identical to avoid loss of inserted genes by recombination or dilute its transcription factors. RESULTS: Based on RNA-seq data, 18 candidate genes with the highest transcription levels at three carbon sources (glucose, glycerol and methanol) were selected and their promoter regions were isolated from GS115 genome. The performance of these promoters on the level of protein production was evaluated using LacZ and EGFP genes as the reporters, respectively. These isolated promoters all exhibited activity to express LacZ gene. Using LacZ as a reporter, of the 18 promoter candidates, 9 promoters showed higher expression levels for the reporter compare to pGAP, a strong promoter widely used for constitutive expression of heterologous proteins in Pichia pastoris. These promoters with high expression levels were further employed to evaluate secreted expression using EGFP as a reporter. 6 promoters exhibited stronger protein expression compare to pGAP. Interestingly, the protein expression driven by pFDH1 was slightly higher than that of commonly used pAOX1 at methanol, and methanol-induced expression of pFDH1 was not repressed by glycerol. CONCLUSION: The various promoters identified in this study could be used for heterologous expression of biosynthetic pathway genes for pharmaceutical or fine chemical production. the methanol-induced pFDH1 that is not repressed by glycerol is an attractive alternative to pAOX1 and may provide a novel way to produce heterologous proteins in Pichia pastoris.