N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.     

Extraabdominal fibromatosis in retroperitoneal space

Background  

Fibromatosis or desmoid tumor covers a broad spectrum of benign fibrous tissue proliferations. It is characterized by infiltrative growth and a tendency towards recurrence; however, unlike sarcoma, it never metastasizes.     

Gene therapy for pancreatic cancer targeting the genomic alterations of tumor suppressor genes using replication-selective oncolytic adenovirus

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we examined the genetic alternations of pancreatic cancer. Based on these results, we are developing a new gene therapy targeting the genetic character of pancreatic cancer using mutant adenoviruses selectively replication-competent in tumor cells. Loss of heterozygosity (LOH) of 30% or more were observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed the significant association with poor prognosis. These data strongly suggest that mutation of the putative suppressor genes, TP53 and SMAD4 play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy targeting tumors without normal TP53 function. E1B-55kDa-deleted adenovirus (AxE1AdB) can selectively replicate in TP53-deficient human tumor cells but not cells with functional TP53. We evaluated the therapeutic effect of this AxE1AdB on pancreatic cancer without normal TP53 function. The growth of human pancreatic tumor in SCID mice model was markedly inhibited by the consecutive injection of AxE1AdB. Furthermore, AxE1AdB is not only the strong weapon but also useful carrier of genes possessing anti-tumor activities as a virus vector specific to tumors without normal TP53 function. It was reported that uracil phosphoribosyl transferase (UPRT) overcomes 5FU resistance. UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits Thymidylate Synthetase (TS). The therapeutic advantage of restricted replication competent adenovirus that expresses UPRT (AxE1AdB-UPRT) was evaluatedin an intra-peritoneal disseminated tumor model. To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the adenovirus, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. These results revealed thatthe AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.     

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura⁺ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys⁻ transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.     

Identification and Mutational Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Sorbitol-Enhanced Glucose-Repressed <Emphasis Type="Italic">srlA</Emphasis> Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.     

Nearest template prediction: a single-sample-based flexible class prediction with confidence assessment

Gene-expression signature-based disease classification and clinical outcome prediction has not been widely introduced in clinical medicine as initially expected, mainly due to the lack of extensive validation needed for its clinical deployment. Obstacles include variable measurement in microarray assay, inconsistent assay platform, analytical requirement for comparable pair of training and test datasets, etc. Furthermore, as medical device helping clinical decision making, the prediction needs to be made for each single patient with a measure of its reliability. To address these issues, there is a need for flexible prediction method less sensitive to difference in experimental and analytical conditions, applicable to each single patient, and providing measure of prediction confidence. The nearest template prediction (NTP) method provides a convenient way to make class prediction with assessment of prediction confidence computed in each single patient's gene-expression data using only a list of signature genes and a test dataset. We demonstrate that the method can be flexibly applied to cross-platform, cross-species, and multiclass predictions without any optimization of analysis parameters.