Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S515

[P-S.11]

Taurine production by systems metabolic engineering ofEscherichia coli

Y.J. Choi 1,∗, J.H. Park 1, S.Y. Lee 1,2

1 Metabolic and Biomolecular Engineering National Research Labora-tory, Department of Chemical and Biomolecular Engineering (BK21Program) and BioProcess Engineering Research Center, KAIST, Daejeon,Repu, Republic of Korea2 Department of Bio and Brain Engineering, and BioinformaticsResearch Center, KAIST, Daejeon, Republic of KoreaKeywords: Taurine; Metabolic engineering; Escherichia coli

Taurine, a free amino acid, has been shown to be essentialin various ways; mammalian development and clinical treatmentsuch as cardiovascular diseases, hepatic disorders, alcoholism andcystic fibrosis. Mammals are able to synthesize taurine but mostmammals have low activity levels of cysteine sulfinic acid decar-boxylase (CSAD), which plays a key role in taurine synthesis and arethus more dependent on dietary supplementation of taurine. Dueto the dependency of taurine uptake on dietary sources, Approx-imately, five to six thousands of tons of taurine are producedannually. However, its production has relied on chemical synthe-sis, which led us to develop a biotechnological method for taurineproduction. Toward this goal, novel synthetic pathway for tau-rine production was established in Escherichia coli, and furthermetabolic engineering was performed. It is the first approach fortaurine production using unique synthetic engineering and it givesa good example of metabolic engineering for the production ofvaluable metabolites. [This work was supported by the Korean Sys-tems Biology Research Project (20090065571) of the Ministry ofEducation, Science and Technology (MEST) through the NationalResearch Foundation of Korea (NRF). Further supports by the WorldClass University Program (R32-2008-000-10142-0) of the MEST,LG Chem Chair Professorship, IBM SUR program, and Microsoft areappreciated.]

doi:10.1016/j.jbiotec.2010.09.818

[P-S.12]

Study of Glial Fibrillary Acidic Protein activity when released byGlioblastoma Multiforme

N. Minari ∗, R. Reviglione, D. Corpillo, A. Giuliano Albo, B. Canepa

LIMA, BioIndustry Park del Canavese S.p.A, Italy

Glial Fibrillary Acidic Protein (GFAP), an intermediate filamentprotein expressed in the cytoskeleton of astrocytes, shares con-siderable structural homology with its family members keratin,desmin and vimentin in the central alpha-helical or rod domain.

GFAP was firsty isolated from the white matter plaques ofpatients with longstanding multiple sclerosis (Eng et al., 2000), nowit is the most widely used marker for cells of astrocytic origin undernormal and pathological conditions.

GFAP is expressed in mature astrocytes, nonmyelinatingSchwann cells, the epithelium of the lens, the epithelial cells of sali-vary glands and their neoplastic cells of mullerian origin. It locates47.8% in nuclear, 39.1% in mitochondrial and 13.0% in cytoplasmiccompartments inside the cell, but is also released from the cell afterbrain stroke and high levels of GFAP are revealed in glioblastomapatient sera (Herrmann et al., 2000; Foerch et al., 2006). However,little is known about its function when released in the extracellularenvironment.

We studied the levels of GFAP released by different cell linesand its activity in various biological assays. The ability of Glioblas-toma Multiforme (GBM) cells (T98G) of releasing GFAP and its levelsrelative to Human Astrocytoma (CCF) and Human Neuroblastoma(SHSY) were verified in conditioned medium through western blot-ting. We revealed GFAP only in T98G medium.

In vitro, GFAP shows proangiogenic activity on endothelial cells.Our hypothesis is an involvement of GFAP in attracting endothelialcells around GBM mass and inducing angiogenesis to contribute totumor supply.

Our aim is to analyse thoroughly this still unknown role ofGFAP, the mechanisms involved and to develop GFAP inhibitingmolecules.

References

Eng, L.F., Ghirnikar, R.S., Lee, Y.L., 2000 Oct. Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem Res. 25 (9–10), 1439–1451.

Herrmann, M., Vos, P., Wunderlich, M.T., de Bruijn, C.H., Lamers, K.J., 2000 Nov.Release of glial tissue-specific proteins after acute stroke: A comparative analy-sis of serum concentrations of protein S-100B and glial fibrillary acidic protein.Stroke 31 (11), 2670–2677.

Foerch, C., Curdt, I., Yan, B., Dvorak, F., Hermans, M., Berkefeld, J., Raabe, A., Neumann-Haefelin, T., Steinmetz, H., Sitzer, M. Serum, 2006 Feb. Glial fibrillary acidicprotein as a biomarker for intracerebral haemorrhage in patients with acutestroke. J Neurol Neurosurg Psychiatry. 77 (2), 181–184, Epub 2005 sep 20.

doi:10.1016/j.jbiotec.2010.09.819

[P-S.13]

Fumaric acid Uptake Mechanism in Saccharomyces cerevisiae:Aerobic vs. Anaerobic Conditions

E. Jamalzadeh ∗, W.M. van Gulik, J.J. Heijnen

TUDelft, NetherlandsKeywords: Fumaric acid; Saccharomyces cerevisiae; Transport

Fumaric acid is a naturally occurring four-carbon dicarboxylicacid that is finding increasing applications in food, pharmaceutical,and polymer industries. Microbial strains such as Rhizopus oryzea,which can produce fumaric acid naturally, cannot tolerate low pHand stoichiometric amount of calcium carbonate should be addedto maintain the neutral pH. This will cause difficulties for the down-stream process and leads to undesired byproducts. An alternative isSaccharomyces cerevisiae, which is considered as a good candidatefor production of such dicarboxylic acids at low pH, which is advan-tageous for the recovery process. This avoids the stoichiometric useof high acid and base and resulting in organic salt production.

Membrane transport of C4-dicarboxylic acids is one of the keyfactors to be examined for such production process improvement.In the production process at low pH, a significant part of secretedfumaric acid is in the undissociated form and this might increasecause back flux of acid into the cells and acidify the cytosol. There-fore, cells need to spend energy to export the protons and importedfumarate, leading to a futile cycle. This decreases the biomass yieldand productivity of fumaric acid.

In this work, we have studied this uptake of fumaric acidby performing different glucose-limited chemostat experimentsin aerobic and anaerobic conditions at different pH and differ-ent sodium fumarate concentrations. We have determined thefumarate uptake rates and interpreted the results using modelsof transport mechanism and kinetics. Experimental results showthat undissociated acid is the transported species since uptake rateincreases by decreasing pH. Moreover, uptake mechanism differsin aerobic and anaerobic conditions. Protein transporters facilitateuptake of undissociated acid in anaerobic conditions while passive