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D D Ryu

Publications and source records attributed to D D Ryu.

At least 19 recordsLinked to original sources
PubMed2000

Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica.

The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.

Cellulase↗
PubMed1998

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene.

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPase. The cloned gene product exhibits 60.3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevesiae PMR1 gene product, a P-type Ca(2+)-ATPase.

ATP-Binding Cassette Transporters↗
PubMed1998

Improvement of heterologous protein productivity using recombinant Yarrowia lipolytica and cyclic fed-batch process strategy.

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).

Bioreactors↗
PubMed1998

Molecular cloning of YlPMR1, a S. cerevisiae PMR1 homologue encoding a novel P-type secretory pathway Ca2+ -ATPase, in the yeast Yarrowia lipolytica.

A novel P-type ATPase gene, Saccharomyces cerevisiae PMR1 homologue (YlPMR1), has been cloned and sequenced in the yeast, Yarrowia lipolytica. The putative gene product has 928 amino acids with a calculated molecular mass of 100050 Da and a pI of 5.15. The deduced amino-acid sequence analysis demonstrated that the cloned gene product contains all 10 of the conserved regions in P-type ATPases and exhibits 55% amino-acid identity to the S. cerevisiae PMR1 gene product; however, it shows a relatively lower homology to PMCA (24%) and SERCA (33%), confirming the presence of a third class of Ca2+-ATPase (secretory pathway Ca2+-ATPase, SPCA). The YlPMR1-disrupted strain shows defective growth in low Ca2+ or EGTA-containing medium. In fact, a longer lag time (60 h) was observed in YlPMR1-defective mutant cells during cultivation in EGTA-containing YPD medium. These growth defects were overcome by adding Ca2+ and Mn2+ into the medium. Interestingly, whereas Mn2+ inhibits growth of the control strain, it significantly improves the growth of YlPMR1-disrupted cells. These results suggest an involvement of the YlPMR1 gene product in Ca2+ and Mn2+ ion homeostasis in Y. lipolytica.

Amino Acid Sequence↗
PubMed1998

Recombinant bioprocess optimization for heterologous protein production using two-stage, cyclic fed-batch culture.

A two-stage, cyclic fed-batch bioprocess was designed, and its performance evaluated to improve rice alpha-amylase productivity by the yeast Yarrowia lipolytica SMY2 (MatA, ade1, ura3, xpr2), ATCC 201847, containing a replicative plasmid coding for a rice alpha-amlyase. Transcription of the recombinant gene is controlled by the XPR2 promoter. The first stage (or growth stage) was operated in the fed-batch mode, and the growth medium, designed to maintain a constant high cell density (i.e., 60 g/l), was fed according to a predetermined and preprogrammed optimal feed rate which, in turn, maintained the specific cell growth rate at an optimal value (i.e., 0.1 h-1). Typically, when the volume in the first stage reached a preset value, a portion of culture broth (i.e., 55%) was transferred to the second stage (or production stage). The remaining cells in the growth stage were then fed with fresh growth medium according to the bioprocess control strategy developed, while induction of alpha-amylase expression and its production was taking place in the second stage. The second stage was also operated in the fed-batch mode, and the production medium designed to maintain a constant high cell density and high productivity of heterologous protein was fed at a predetermined and preprogrammed rate, which maintained the specific cell growth rate at an optimal level. The volumetric alpha-amylase productivity achieved (1835 units l-1 h-1) from the two-stage, cyclic fed-batch culture process was twofold higher than that of the fed-batch culture process. The genetic stability of the recombinant strain and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-stage, cyclic fed-batch process for production of heterologous protein.

Recombinant Proteins↗
PubMed1998

Disruption of PMR1, encoding a Ca2+-ATPase homolog in Yarrowia lipolytica, affects secretion and processing of homologous and heterologous proteins.

The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca2+-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the YlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca2+-ATPase genes on the secretion of rice alpha-amylase was also observed in the Y. lipolytica and S. cerevisiae PMR1-disrupted mutants. Unlike rice alpha-amylase, the secretion of Trichoderma reesei endoglucanase I (EGI) was not influenced by the YlPMR1 disruption. However, the secreted EGI from the YlPMR1-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant. Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.

ATP-Binding Cassette Transporters↗
PubMed1997

Expression, secretion, and processing of rice alpha-amylase in the yeast Yarrowia lipolytica.

The gene encoding rice alpha-amylase in Oryza sativa was expressed in the yeast Yarrowia lipolytica, which is a potential host system for heterologous protein expression. For efficient secretion, the strong and inducible XPR2 promoter was used in the construction of four kinds of expression vectors with the following configurations between the XPR2 promoter and terminator: 1) XPR2 prepro-region-rice alpha-amylase coding sequence, 2) rice alpha-amylase signal peptide-rice alpha-amylase coding sequence, 3) XPR2 signal peptide-rice alpha-amylase coding sequence, and 4) XPR2 signal peptide-dipeptide stretch-rice alpha-amylase coding sequence. Secretion of active recombinant rice alpha-amylase into the culture medium was achieved only in the first two cases, demonstrating that the XPR2 signal peptide is not sufficient to direct the secretion of heterologous protein. Furthermore, our study shows that the XPR2 prepro-region causes imprecise processing (after Pro150-Ala151 or Val135-Leu136 instead of Lys156-Arg157) and leads to N-terminal amino acid sequences that differ from that of native rice alpha-amylase. Secondary structure analysis proposed that the structural form in the vicinity of the KEX2-like endopeptidase processing site in the XPR2 pro-region might play a critical role in the processing of heterologous proteins. These results suggest that the XPR2 pro-region is dispensable for obtaining the precise N-terminal amino acid in heterologous protein secretion. In contrast, utilizing the rice alpha-amylase signal peptide was sufficient in directing secretion of recombinant protein with the expected N-terminal sequence, indicating that the signal peptide of rice alpha-amylase was effectively recognized and processed by the Y. lipolytica secretory pathway.

Cloning, Molecular↗
PubMed1990

Membrane resistance to oxygen transport inside hybridoma cells in suspension culture.

We have examined oxygen transport from media to a single hybridoma cell for different dissolved oxygen concentrations in media and oxygen consumption rates of cells. Intracellular oxygen concentration profiles inside the cell were simulated using currently available parameters. From the simulation, it was found that mass transfer resistance at the boundary layer does not seem to be significant. Intracellular oxygen concentrations did not differ very much from outside media concentration and the concentration gradients were relatively low for all five different media DOC's (from 0.1 to 100% air saturation). From these results, it is suspected that either hybridoma cells have better tolerance to high intracellular oxygen concentrations than normal cells or that the cell membrane has a certain mechanism for limiting oxygen transport into the cell to avoid the oxidative damage.

Animals↗
PubMed1990

Effect of intercalating dyes on the production of antibiotics by Micromonospora rosaria and Micromonospora purpurea.

The effect of treatment with various intercalating dyes on the ability to produce antibiotics in Micromonospora rosaria and Micromonospora purpurea was studied. Treatment with acriflavine resulted in a high frequency loss of antibiotic productivity in both species. In M. rosaria, the loss of antibiotic-producing ability appeared to be strain-dependent. In M. purpurea, up to 90% of colonies were found to have lost gentamicin-producing ability when protoplasts were used in the test. These antibiotic-nonproducing strains were further studied. The following observations were made: (1) Unlike the producing ability, the resistance to the antibiotics is a very stable character in both species. (2) Protoplast fusion analysis indicates that rosamicin-nonproducing characteristics of MR 217-AF2 and MR 217-AF3 strains induced by the acriflavine treatment is due to chromosomal mutation or rearrangement but not to loss of a plasmid. (3) Gentamicin-nonproducing strains of M. purpurea responded differently to the supplementation of streptamine or DOS in the culture medium. When supplemented with streptamine or DOS, some of these strains regained the ability to produce antibiotic, showing that the biosynthesis of intermediate was affected in these strains.

Anti-Bacterial Agents↗
PubMed1989

Structured modeling and simulation of oxygen transport to hybridoma cell in a suspension culture: theoretical analysis.

The importance of the consideration of micro-anatomical and cytological observation for the oxygen utilization by hybridoma cells are emphasized. Tumor cells have considerably lower content of mitochondria and that cells metabolize much of their carbon source by anaerobic pathway, therefore, consumes much less oxygen. Because of the fact that hybridoma cells are derived from a myeloma cell, which is a type of tumor cell, there may be possibility that the oxygen demand could be closely related to mitochondrial content of the resulting cell. Possible correlation between the optimum DOT and mitochondrial content of the hybridoma cells is hypothesized. By simulating oxygen transport inside of a single hybridoma cells for the cases of three different mitochondrial contents, possible differences in the optimum DOT according to the mitochondrial contents were studied and the results reported.

Animals↗
PubMed1985

Relationship between butirosin biosynthesis and sporulation in Bacillus circulans.

The relationship between butirosin biosynthesis and certain biochemical characteristics related to sporulation in a strain of Bacillus circulans NRRL B-3313 was examined. The cellular content of dipicolinic acid increased while the amount of poly-beta-hydroxybutyrate decreased with changes in antibiotic productivity. Oligosporogenous mutants failed to synthesize the antibiotic and to degrade poly-beta-hydroxybutyrate. These observations suggest that spore formation may be related to antibiotic production in this strain of B. circulans.

Anti-Bacterial Agents↗
PubMed1984

An improved synthesis of captopril.

An improved synthesis of captopril using methacrylic acid as the starting material is described. Treatment of methacrylic acid (I) with a hydrogen halide gave the 3-halogeno-2-methylpropanoic acids II and III, which were treated with thionyl chloride to yield the corresponding 3-halogeno-2-methylpropanoyl chlorides IV and V. Treatment of IV or V with L-proline yielded the N-(R,S-3-halogeno-2-methylpropanoyl)-L-prolines VI and VII, which were separated into optically pure R- and S-diastereoisomers using dicyclohexylamine. Treatment of halides of VI or VII with methanolic ammonium hydrosulfide gave captopril in 28% yield.

Captopril↗
PubMed1984

Enzymatic synthesis of phenoxymethylpenicillin using Erwinia aroideae enzyme.

Enzymatic synthesis of phenoxymethylpenicillin from 6-aminopenicillanic acid and phenoxyacetic acid methyl ester was attempted by using partially purified alpha-acylamino-beta-lactam acylhydrolase I (ALAHase I) enzyme from Erwinia aroideae NRRL B-138. The reaction rates were carefully followed by determination of 6-aminopenicillanic acid (6-APA), phenoxymethylpenicillin (PNV), phenoxyacetic acid (POA), phenoxyacetic acid methyl ester (POM), and phenoxyacetylglycine (POG) using high performance liquid chromatography. Among the acyl donors tested, POM gave the highest yield (12.2% based on 6-APA). The overall conversion increased almost linearly with an increase in molar ratio of POM to 6-APA up to 4:1. The effects of organic solvents on the overall yield were also evaluated. Some improvement of PNV yield was observed when ethanol, 2-propanol, and acetone were used. ALAHase I was found to carry out three reactions simultaneously: transfer of acyl group to acyl acceptor to form semisynthetic beta-lactam antibiotic; hydrolysis of acyl donor in amide or ester bond, and hydrolysis of semisynthetic beta-lactam antibiotic which was produced by the enzyme. It was also observed that the hydrolysis reactions of POM and PNV were irreversible in this reaction system. The optimal pH for the three reactions was different. They were: pH 9.0 for POM hydrolysis, 6.8 for the transfer of phenoxyacetyl group to 6-APA, and 6.0 for the PNV hydrolysis. The apparent Km values for POM, 6-APA and PNV were estimated as 33, 25 and 31 mM, respectively.

Erwinia↗
PubMed1983

Genetic recombination in Micromonospora rosaria by protoplast fusion.

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.

Genes, Bacterial↗