Seong-Joo Hong

A novel prepurification for paclitaxel from plant cell cultures

A novel prepurification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. This method was a simple and efficient procedure, for the isolation and prepurification of paclitaxel from the biomass of Taxus chinensis, consisting of active clay treatment, followed by two steps of precipitation. The use of active clay treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimize solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. This process is readily scalable to pilot plant scale and eventually to a production environment where multikilogram quantities of material are expected to be produced.

Evaluation of central metabolism based on a genomic database ofSynechocystis PCC6803

Cyanobacteria produce industrially important secondary metabolites such as lipopeptide, oligosaccharide, fatty acid (esp. sulfolipid),etc. Among them,Synechocystis PCC6803 is the first strain with a publicly available full genome sequence, as of 1996, and is one of the most extensively studied photosynthetic microorganisms. Using this genomic information, the central metabolism ofSynechocystis PCC6803 was reconstructed, including photosynthesis, oxidative phosphorylation, glycolysis, pyruvate metabolism, TCA cycle, carbon fixation, and transport system. Each biochemical reaction was carefully incorporated into the model, taking into consideration the metabolite formula, stoichiometry, charge balance, and thermodynamic properties using information from genomic and metabolic databases as well as biochemical literature. The metabolic flux of the model was calculated using flux balance analysis according to its cultivation with various carbon sources. The results of simulation were in accordance with experimental data, which suggests that the central metabolism model can properly estimate the behavior ofSynechocystis PCC6803. This model would aid in the understanding of the whole cell metabolism ofSynechocystis PCC6803, the first effort of its kind for photosynthetic bacteria.

Effects of Light Intensity and Nitrogen Starvation on Glycerolipid, Glycerophospholipid, and Carotenoid Composition in Dunaliella tertiolecta Culture

Time-course variation of lipid and carotenoid production under high light (300 μE/m2s) and nitrogen starvation conditions was determined in a Dunaliella tertiolecta strain. Nanoelectrospray (nanoESI) chip based direct infusion was used for lipid analysis and ultra-performance liquid chromatography (UPLC) coupled with a photodiode array (PDA) or atmospheric chemical ionization mass spectrometry (APCI-MS) was used for carotenoid analysis. A total of 29 lipids and 7 carotenoids were detected. Alterations to diacylglyceryltrimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG) species were significant observations under stress conditions. Their role in relation to the regulation of photosynthesis under stress condition is discussed in this study. The total carotenoid content was decreased under stress conditions, while ã-carotene was increased under nitrate-deficient cultivation. The highest productivity of carotenoid was attained under high light and nitrate sufficiency (HLNS) condition, which result from the highest level of biomass under HLNS. When stress was induced at stationary phase, the substantial changes to the lipid composition occurred, and the higher carotenoid content and productivity were exhibited. This is the first report to investigate the variation of lipids, including glycerolipid, glycerophospholipid, and carotenoid in D. tertiolecta in response to stress conditions using lipidomics tools.

Elucidation of the growth delimitation of Dunaliella tertiolecta under nitrogen stress by integrating transcriptome and peptidome analysis

Dunaliella tertiolecta LB 999 is an oleaginous microalgae species that produces large quantities of lipid and starch during nitrogen starvation; however, nitrogen starvation also limits the cell growth. In order to understand the underlying mechanisms of this phenomenon, the transcriptome and peptidome of D. tertiolecta LB 999 grown under different nitrogen and light conditions were analyzed. Integration of the de novo assembly of transcriptome sequencing reads with peptidome analysis revealed 13,861 protein-coding transcripts, including 33 transcripts whose expression patterns were significantly altered along with the growth phenotypes. Interestingly, 21 of these genes, which were highly enriched in the plastid region, were associated with chlorophyll synthesis and tetrahydrofolate-mediated C1 metabolism. Furthermore, intracellular glutamate levels are predicted to be the main factor that acts as a switch for the regulation of cell growth and carbon accumulation. These data provide the genetic information of D. tertiolecta for its future applications.

Effect of Ethephon as an Ethylene-Releasing Compound on the Metabolic Profile of Chlorella vulgaris.

In this study, Chlorella vulgaris (C. vulgaris) was treated with ethephon at low (50 μM) and high (200 μM) concentrations in medium and harvested at 0, 7, and 14 days, respectively. The presence of ethephon led to significant metabolic changes in C. vulgaris, with significantly higher levels of α-tocopherol, γ-aminobutyric acid (GABA), asparagine, and proline, but lower levels of glycine, citrate, and galactose relative to control. Ethephon induced increases in saturated fatty acids but decreases in unsaturated fatty acids. The levels of highly saturated sulfoquinovosyldiacylglycerol species and palmitic acid bound phospholipids were increased on day 7 of ethephon treatment. Among the metabolites, the productivities of α-tocopherol (0.70 μg/L/day) and GABA (1.90 μg/L/day) were highest for 50 and 200 μM ethephon on day 7, respectively. We propose that ethephon treatment involves various metabolic processes in C. vulgaris and can be an efficient way to enrich the contents of α-tocopherol and GABA.

Label-free non-invasive quantitative measurement of lipid contents in individual microalgal cells using refractive index tomography

Microalgae are promising candidates for biofuel production due to their high lipid content. To facilitate utilization of the microalgae for biofuel, rapid quantification of the lipid contents in microalgae is necessary. However, conventional methods based on the chemical extraction of lipids require a time-consuming destructive extraction process. Here, we demonstrate label-free, non-invasive, rapid quantification of the lipid contents in individual micro-algal cells measuring the three-dimensional refractive index tomograms. We measure three-dimensional refractive index distributions within Nannochloropsis oculata cells and find that lipid droplets are identifiable in tomograms by their high refractive index. In addition, we alter N. oculata under nitrogen deficiency by measuring the volume, lipid weight, and dry cell weight of individual cells. Characterization of individual cells allows correlative analysis between the lipid content and size of individual cells.

Comparative lipidomic profiling of two Dunaliella tertiolecta strains with different growth temperatures under nitrate-deficient conditions.

The metabolic changes that occur in Dunaliella tertiolecta upon exposure to low temperatures and nitrate deficiency were analyzed by exploring the fatty acid composition and lipid profile of two strains that were acclimated to different temperatures. The results indicate that the levels of linolenic acid (C18:3) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) were significantly higher in the low-temperature (15 °C) strain (SCCAP K-0591) than in a strain grown at 21 °C (UTEX LB999). In addition, DGTS accumulated in LB999 under nitrate-deficient conditions, while the levels of most lipids, including DGTS, remained almost consistent in K-0591. The higher levels of DGTS in K-0591 suggest that DGTS could play a role in adaptation to low temperatures and nitrate deficiency in this organism. The results of this research could be applied to the development of new microalgal strains with tolerance of low temperature and nitrate deficiency by metabolic engineering targeted to DGTS species.

Microalgal Systems Biology Through Genome-Scale Metabolic Reconstructions for Industrial Applications

Abstract Since the publication of the complete genome sequence of Synechocystis sp. PCC6803, an enormous amount of genomic data for microalgae has been made available on the web. As of November 2013, genome information for 39 cyanobacteria and 24 eukaryotic microalgae is publicly accessible. Additionally, the development of high-throughput omics technologies has driven the accumulation of information on microalgal metabolism. With the evolution of omics technologies, systems biology has become a tool for processing massive amounts of omics data; it has revealed global microalgal metabolic networks and enhanced our understanding of cellular physiology and regulation. Using in silico models of microalgae generated by systems biology, researchers can investigate the photosynthetic pathways that convert inorganic carbon to organic carbon and cellular responses to environmental changes. Such studies will provide opportunities for enhancing microalgal biomass and productivity.

Enhanced Production of Fatty Acids via Redirection of Carbon Flux in Marine Microalga Tetraselmis sp.

Lipids in microalgae are energy-rich compounds and considered as an attractive feedstock for biodiesel production. To redirect carbon flux from competing pathways to the fatty acid synthesis pathway of Tetraselmis sp., we used three types of chemical inhibitors that can block the starch synthesis pathway or photorespiration, under nitrogen-sufficient and nitrogen-deficient conditions. The starch synthesis pathway in chloroplasts and the cytosol can be inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 1,2-cyclohexane diamine tetraacetic acid (CDTA), respectively. Degradation of glycine into ammonia during photorespiration was blocked by aminooxyacetate (AOA) to maintain biomass concentration. Inhibition of starch synthesis pathways in the cytosol by CDTA increased fatty acid productivity by 27% under nitrogen deficiency, whereas the blocking of photorespiration in mitochondria by AOA was increased by 35% under nitrogen-sufficient conditions. The results of this study indicate that blocking starch or photorespiration pathways may redirect the carbon flux to fatty acid synthesis.

Microalgal Systems Biology for Biofuel Production

In recent years, microalgae has received a lot of attention as potential sources of renewable energy, especially given the increase in oil prices and environmental concerns. Therefore, microalgal systems biology can shed light on complex interactions in biological systems through integration of various omics data. Genome-scale metabolic reconstruction can provide insights into cellular metabolism and species-specific adaptive features, whereas in silico analysis is a powerful tool for analysis of metabolic flux and identification of gene targets for enhanced production of biofuel precursors. Here, we highlight the current state of research in microalgal systems biology and evaluate the potential for future sustainable microalgae-based biofuel engineering and development of “green cell factories.”