Biomedical subjects

Sang Yup Lee

Publications and source records attributed to Sang Yup Lee.

9 recordsLinked to original sources

PubMed2003

In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli.

The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild-type E. coli and recombinant E. coli producing poly(3-hydroxybutyrate) [P(3HB)]. The flux of acetyl-CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production. It was notable to find from in silico analysis that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB)-accumulating conditions. To prove the role of ED pathway on P(3HB) production, a mutant E. coli strain, KEDA, which is defective in the activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon. The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4). The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co-expression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis.

Alcaligenes↗

PubMed2003

Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture.

Combined transcriptome and proteome analysis was carried out to understand metabolic and physiological changes of Escherichia coli during the high cell density cultivation (HCDC). The expression of genes of TCA cycle enzymes, NADH dehydrogenase and ATPase, was up-regulated during the exponential fed-batch period and was down-regulated afterward. However, expression of most of the genes involved in glycolysis and pentose phosphate pathway was up-regulated at the stationary phase. The expression of most of amino acid biosynthesis genes was down-regulated as cell density increased, which seems to be the major reason for the reduced specific productivity of recombinant proteins during HCDC. The expression of chaperone genes increased with cell density, suggesting that the high cell density condition itself can be stressful to the cells. Severe competition for oxygen at high cell density seemed to make cells use cytochrome bd, which is less efficient but has a high oxygen affinity than cytochrome bo(3). Population cell density itself strongly affected the expression of porin protein genes, especially ompF, and hence the permeability of the outer membrane. Expression of phosphate starvation genes was most strongly up-regulated toward the end of cultivation. It was also found that sigma(E) (rpoE) plays a more important role than sigma(S) (rpoS) at the stationary phase of HCDC. These findings should be invaluable in designing metabolic engineering and fermentation strategies for the production of recombinant proteins and metabolites by HCDC of E. coli.

Amino Acid Sequence↗

PubMed2003

Microbial cell-surface display.

Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sandwich fusion. The characteristics of carrier protein, passenger protein and host cell, and fusion method all affect the efficiency of surface display of proteins. Microbial cell-surface display has many potential applications, including live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.

Adsorption↗

PubMed2003

Enhanced production of recombinant proteins in Escherichia coli by filamentation suppression.

During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.

Bacterial Proteins↗

PubMed2003

Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids.

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.

Carboxylic Ester Hydrolases↗

PubMed2003

Enhanced production of insulin-like growth factor I fusion protein in Escherichia coli by coexpression of the down-regulated genes identified by transcriptome profiling.

The transcriptome profiles of recombinant Escherichia coli producing human insulin-like growth factor I fusion protein (IGF-I(f)) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-I(f) after induction. Among these down-regulated genes, we rationally selected and coexpressed in E. coli producing IGF-I(f) the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-I(f) could be increased from 1.8 +/- 0.13 (+/- standard deviation) to 4.3 +/- 0.24 g/liter. The volumetric productivity was also increased from 0.36 +/- 0.027 to 0.82 +/- 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.

Aquaporins↗

PubMed2003

Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli.

The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional beta-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.

Acyl Coenzyme A↗

PubMed2002

Metabolic engineering of Escherichia coli for the production of medium-chain-length polyhydroxyalkanoates rich in specific monomers.

The Escherichia coli fabG(Ec) gene and the Pseudomonas aeruginosa rhlG(Pa) gene, which encode 3-ketoacyl-acyl carrier protein reductase, were expressed in E. coli W3110 and its fadA mutant strain WA101 to examine their roles in medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis from fatty acids. When one of these 3-ketoacyl-acyl carrier protein reductase genes was co-expressed with the Pseudomonas sp. 61-3 PHA synthase gene (phaC2(Ps)) in E. coli W3110, MCL-PHA composed mainly of 3-hydroxyoctanoate and 3-hydroxydecanoate was synthesized from sodium decanoate. When the fabG(Ec) gene and the phaC2(Ps) gene were co-expressed in the fadA mutant E. coli strain WA101, MCL-PHA rich in 3-hydroxydecanoate monomer up to 93 mol% was accumulated from sodium decanoate. This was possible by efficiently redirecting 3-ketoacyl-coenzymes A from the beta-oxidation pathway to the PHA biosynthesis pathway without losing two carbon units, the strategy of which can be extended for the production of MCL-PHAs rich in other specific monomers.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗

PubMed2002

Excretion of human beta-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli.

Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E. coli MBEL-BL101. For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained.

Bacterial Outer Membrane Proteins↗