Jungoh Ahn

Genome-scale metabolic reconstruction and in silico analysis of methylotrophic yeast Pichia pastoris for strain improvement

BackgroundPichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeasts metabolism.ResultsA fully compartmentalized metabolic model of P. pastoris (iPP 668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.ConclusionThe genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

Treatment of spontaneous arterial dissections with stent placement for preservation of the parent artery.

SummaryBackground. A wide variety of treatment regimens have been advocated for dissections involving the intracranial arteries. Recently, the stent can be used to exclude the aneurysm from the circulation and preserve the parent artery. We evaluated the safety and efficacy of stent angioplasty for intracranial arterial dissections.Methods. Ten patients with spontaneous dissections, nine vertebral artery and one internal carotid artery lesions underwent endovascular treatment using stent placement as primary treatment modality. One stent placement was attempted in five patients initially. Three patients were intentionally treated with two overlapping stents which completely covered the aneurysm orifice. Two tandem stents were used in one patient to allow spanning the entire length of the dissection. Stent-assisted coil embolization was performed in one patient.Results. Of the 10 patients in whom stenting was tried, the overall success in reaching the target lesion with stents was 90%. Of the 9 patients treated with stents, stent release and positioning were considered optimal in 7 patients (77.8%) and suboptimal in two. Lesions of 8 patients were improved or stable in angiographic follow-up. However, one pseudo-aneurysm was enlarged, and subsequently, was treated by proximal occlusion using coils. There were no instances of postprocedural ischaemic attacks, new neurological deficits, and no new minor or major strokes prior to patient discharge. All parent arteries of the patient who underwent the successful procedure were preserved. On the modified Rankin scale used for the follow up, all patients were assessed as functionally improved or of stable clinical status.Conclusions. The success in reducing dissection-induced stenosis or pseudo-aneurysm, the patency rate obtained at follow-up, and the lack of strokes (ischaemic or haemorrhagic) suggest that stent placement offers a viable alternative to complex surgical procedures or deconstructive procedures. The long-term efficacy and durability of stent placement for arterial dissection remains to be determined in a large series.

How to overcome the limitations to determine the resection margin of pituitary tumours with low-field intra-operative MRI during trans-sphenoidal surgery: usefulness of Gadolinium-soaked cotton pledgets.

SummaryObjective. Intra-operative MRI (iMRI) is used as an immediate intra-operative quality control, allowing surgeons to extend resections in situations involving residual tumour remnants. Despite these advantages, low-field iMRI has some limitations with regards to image quality and artefacts. The aim of this study is to report our experience with bone wax and Gadolinium-soaked cotton pledgets in obtaining precise tumour resection margins using low-field iMRI. Patients and methods. The study group included 63 consecutive patients who underwent endonasal trans-sphenoidal surgery with use of intra-operative low-field iMRI (0.15 T, PoleStar N20, Medtronic Navigation, Louisville, CO, U.S.A.). The indications for intra-operative MRI use included a suprasellar or retrosellar extension (n = 23), cavernous sinus invasion (n = 21), a tumour located in the vicinity of critical anatomic structures (such as the internal carotid artery, n = 10), recurrent or revision procedures (n = 5), and pre-operative imaging revealing unusual anatomy (n = 4). Results. Overall, among the 51 patients with intended complete tumour removal, iMRI revealed definite tumour remnants or suspicious findings in 13 patients (25.5%), leading to an extended resection and allowing completion of the resection in 10 patients. There was an increased rate of complete tumour removal from 74.5% (38 out of 51) to 94.1% (48 out of 51). The iMRI scan for complete tumour removal was more efficient in the group receiving Gadolinium-soaked cotton pledgets (85.2–100%) than in the group receiving bone wax or the conventional method (62.5–87.5%). The results of iMRI and the estimation by the surgeon concerning the extent of resection revealed a discrepancy in five patients (15.6%) in the Gadolinium-soaked cotton pledgets application group, and in 14 (45.2%) of the bone wax application group. Conclusions. More valuable information for determining the resection margin can be obtained with the use of contrast-soaked cottonoid packing in the tumour resection cavity during iMRI scanning. We believe that the use of this simple method reduces the false-positive results and also overcomes the disadvantages of low-field iMRI.

Tumor Tissue Identification in the Pseudocapsule of Pituitary Adenoma: Should the Pseudocapsule be Removed for Total Resection of Pituitary Adenoma?

OBJECTIVE The microsurgical pseudocapsule can be found in the transition zone between an adenoma and the surrounding normal pituitary tissue. We investigated the precise histology of the pseudocapsule. Furthermore, we evaluated the remission rate, the changes in pituitary function, and the recurrence rate after intensive resection of the pseudocapsule. METHODS In 616 patients with pituitary adenomas (Hardy Types I–III) over a period of 14 years, we introduced intensive resection of the microsurgical pseudocapsule to achieve complete tumor removal. A combined pituitary function test and radiological study were performed on the patients before surgery, 1 year after surgery, and at subsequent 1.5-year intervals 2 to 13 years postoperatively. RESULTS Microsurgical pseudocapsules were identified in 343 (55.7%) of 616 patients, and the distinct microsurgical pseudocapsules were observed in 180 (52.5%) of these patients. In the remaining 163 patients, the microsurgical pseudocapsules were incompletely developed. Tumor cluster infiltration was present in the pseudocapsule in 71 (43.6%) of these patients. Aggressive resection of the microsurgical pseudocapsule was more often required in larger tumors than in smaller ones. The presence of a pseudocapsule was slightly more frequent in prolactin-secreting tumors (70.9%) than in growth hormone-secreting (55.0%) and adrenocorticotropic hormone-secreting (40.0%) tumors. In the 243 patients of the total resection group who underwent combined pituitary function tests more than 2 times after surgery, the surgical remission rate was 99.1% in clinically nonfunctional tumors, 88% in growth hormone-secreting, 70.6% in prolactin-secreting, and 100% in adrenocorticotropic hormone-secreting tumors. The surgical remission rate was 86.2% in the presence of a pseudocapsule and 94.3% in the absence of a pseudocapsule. Preoperative hypopituitarism improved in 140 patients (57.6%), persisted in 47 patients (19.3%), and was aggravated in 33 patients (13.6%). There was no statistical difference in improvement or deterioration of pituitary function according to the existence or absence of the pseudocapsule. The tumor recurrence rate was 0.8% in the total resection group and was 42.1% in the subtotal resection group. CONCLUSION We have shown that tumor tissue is frequently present within the pseudocapsule, suggesting that any tumor remnant in the pseudocapsule could be a source of recurrence and an obstacle to achieving complete remission. These results indicate that intensive resection of the pseudocapsule could result in a higher remission rate without deteriorating pituitary function.

Pituitary adenoma and concomitant Rathke’s cleft cyst

SummaryAlthough pituitary adenomas and Rathke’s cleft cysts have a shared ancestry, they rarely occur simultaneously. Only 32 reports involving a pituitary adenoma and a concomitant Rathke’s cleft cyst were identified upon review of the literature. Most initial presenting complaints include hormonal symptoms, visual disturbances, and headache. Next to growth hormone, Prolactin was the most commonly hypersecreted pituitary hormone. Rathke’s cleft cysts show variable position, size, and signal intensity on magnetic resonance imaging (MRI).Here, we report a patient with a growth hormone- secreting pituitary adenoma associated with a Rathke’s cleft cyst. The mass contained two different signal intensities on MRI. The lesion was successfully removed assisted by intraoperative MRI, when the presence of both lesions was confirmed.When a non-enhancing cyst-like structure is demonstrated on imaging in a patient with a pituitary adenoma, the possibility of a coexisting Rathke’s cleft cyst should be considered.

Identification of novel immunogenic proteins in pathogenic Haemophilus parasuis based on genome sequence analysis

Haemophilus parasuis causes contagious porcine Glässers disease, which is occurring worldwide and leads to severe losses in the pig industry. To identify novel antigen candidates against this disease, 22 surface-exposed or secreted proteins were selected from the annotated H. parasuis genome by reverse vaccinology strategy. Expression of these proteins in Escherichia coli was attempted. Immunogenicity of the expressed candidates was assessed using Western blot analysis with mouse-derived antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5. Three ABC-type transporters (OppA, YfeA and PlpA) and 1 curli protein assembly (CsgG) were identified as potent immunogenic proteins. The proteins show cross-reactions when tested with sera raised against serovars 4 and 5 of H. parasuis.

Enhancement of monascus pigment production by the culture of Monascus sp. J101 at low temperature

In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 °C. In a batch culture at 30 °C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 °C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 °C was 10 times greater than at 30 °C.

Evaluation of a silica-coated magnetic nanoparticle for the immobilization of a His-tagged lipase

Magnetic particles of size 10 nm have been coated with silica to a mean diameter of 40 nm and charged with Cu2+ ions via a multidentate ligand, iminodiacetic acid (IDA), for the immobilization of His-tagged Bacillus stearothermopilus L1 lipase. Microporous (average pore diameter of 60 Å) silica gel with a mean particle diameter of 115 µm has been used as a comparative support material. The molar ratio of Cu2+ to IDA was found to be 1:1.14 and 1:1.99 in the silica gel and the silica-coated magnetic nanoparticles (SiMNs), respectively. The specific activity of the immobilized enzyme was found to conform to the following order: Cu2+-charged SiMN>SiMN>Cu2+-charged silica gel>silica gel. When it was immobilized on the Cu2+-charged SiMNs, over 70% of the initial activity of the lipase remained after it had been reused five times. However, only 20% of the initial activity remained after the enzyme immobilized on the Cu2+-charged silica gel had been reused five times. For the enzyme immobilized on supports without Cu2+ cations, all activity was lost after threefold reuse. The differences in the specific activities and the efficiencies of reuse of the enzymes immobilized on the various support materials are discussed in terms of immobilization mechanisms (physical adsorption vs. coordination bonding), mass transfer of a substrate and a product of the enzyme reaction, and the status of the Cu (Cu bound to the IDA on the silica layer vs. Cu directly adsorbed on the silica layer).

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

ABSTRACT To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na+)-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (PPHO89) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. PPHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1α and the glyceraldehyde-3-phosphate dehydrogenase promoter, PPHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple PPHO89-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of PPHO89 for controlled production of recombinant proteins in P. pastoris.

Gamma-Aminobutyric Acid Production Using Immobilized Glutamate Decarboxylase Followed by Downstream Processing with Cation Exchange Chromatography

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.