Natural flavor and fragrance compounds are of great value to the food, cosmetics, and medical industries. The price of these natural chemicals can be extremely high due to their low concentration in the plant material. Many herbs and spices contain high concentrations of essential oils that resemble desirable flavor and aroma compounds. By using a single enzymatic step, it is possible to convert these abundant essential oils into natural flavor and aroma chemicals. The overall objectives of the research is to cultivate and screen for plants with a high content of specific compounds in essential oils, and to develop a biotransformation process for the production of natural flavors and fragrances of high commercial value. The research plan includes the following parts: Hybridization and introduction of plants containing essential oils suited for bioconversion Developing screening techniques for microorganisms capable of converting substances such as: eugenol and isoeugenol, methyl cinnamate, trans-anethole, isosafrole, and valencene, into vanillin, benzaldehyde, anisaldehyde, heliotropin, and nootkatone, respectively. Characterizing the enzymatic pathways involved in the bioconversions
1) Cloning, sequencing, and over-producing the transforming enzymes
2) Scaling up a biotransformation process.
A successful development of this research will increase the value of many common herbs and will facilitate the economic production of natural flavors and fragrances.

Shimoni, E., U. Ravid, and Y. Shoham. Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin. J. Biotechnol 78:1-9, 2000.

Lewinson, E., I. Ziv-Raz, N. Dudai, Y. Tadmor, E. Lastochkin, O. Larkov, D. Chaimovitsh, U. Ravid, E. Putievsky, E. Pichersky, and Y. Shoham. Biosysnthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L): Developmental and cghemotypic association of allylphenol O-methyltransferase activities. Plant Science 160:27-35, 2000.

Shimoni Eyal, Timor Baasov, Uzi Ravid, and Yuval Shoham. The trans-anethole degradation pathway in an Arthrobacter sp. J. Biol. Chem. 277:11866-11872, 2002

The ability of E. coli chaperonin GroEL complexes to assist in correct folding of polypeptides is based on their ability to selectively bind polypeptide folding intermediates in non-native or mis-folded conformations, and to mediate their correct folding through cycles of binding and release. Nevertheless, little is known about the restoration activity of chaperonins since most of the in vitro data collected until now is from refolding experiments, in which the denatured polypeptide is diluted in a refolding solution that already contained excess amounts of chaperonin complex which bind very early folding intermediates. In order to further understand the extent of the E. coli chaperonin GroEL "proof-reading" activity (i.e., identifying rather late mis-folded intermediates and bringing them back into the correct folding pathway), we have tested the ability of GroEL to act in unfolding that restores the activity of "dead-end" folding conformations. We chose to follow the spontaneous in vitro refolding of the heterodimeric Vibrio harveyi bacterial luciferase. Our study demonstrates that GroEL does not only protect polypeptides against aggregation, but can also rescue polypeptides which have undergone early steps of aggregation. This was done by dissolving the aggregates and unfolding them in order to allow correct folding and assembly.

Gancz, H., Lavie, O., Kessel, M., Cohen, Y., Kashi, Y. Dissociation of protein aggregates by the chaperonin GroEL In Conformational Diseases - A Compendium/ Beka Solomon, Albert Taraboulos and Ephraim Katchalski-Katzir. The Center for the Study of Emerging Diseases. Chapter 19: 159-169, 2001.

The eukaryotic Hsp60 cytoplasmatic chaperonin CCT (Chaperonin Containing TCP1 complex) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTa and CCTb, in yeast Sacchromyces cerevisiae. Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study mRNA and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3 to 4-fold increase in mRNA levels of CCTa and CCTb genes after cold shock at 48C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTa protein, which is expressed at 108C, but not at 48C. Transfer of 48C cold shocked cells to 108C induced a 5-fold increase in the CCTa protein level. By means of fluorescent immuno-staining and confocal microscopy, we found CCTa to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTa was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTa protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and transition to growth at higher temperatures, as found for other HSPs during the recovery phase from heat shock.

Somer L., Marcovici O., Dror T., Hashmueli S., Kashi Y. The Eukaryote Chaperonin CCT is a cold shock protein in Sacchromyces cerevisiae. Cell Stress & Chaperones (7) no. 1: 47-54, 2002.

During viral infection the host cell machinery is committed to the production of new virus particles. In addition to transcription, translation and replication, the protein folding machinery is also engaged. Our preliminary study has indicated that the eukaryotic cytosolic chaperon - CCT (Chaperonin Containing TCP1 ) may be used for folding viral proteins. This is a unique aspect of the viral life cycle. The study entails the formation of Ty1- Virus-Like Particles (VLPs) in Saccharomyces cerevisiae that serve as a general model for the function of CCT in retroviral life cycle, given the similarity between human retroviral infection and behavior of the yeast S.cerevisiae Ty1 retrotransposon elements.
Our study indicates that the formation of Ty1-VLPs and transposition of Ty1 elements requires the chaperonin CCT complex. It explored the mechanism of CCT-mediated protein folding and oligomeric assembly in eukaryotes and it’s function in the viral life cycle.

Our research is focused on two major aspects.
1) Production of large quantities of biological and biomedical compounds
2) The study of the baculovirus based expression system.
Our major achievements are:
1) Production of large quantities of biological and biomedical compounds.
In collaboration with Prof. Yuval Shoham we haveoptimized protein production by the baculovirus expression system in shake flasks. This enabled us to produce large amounts of biologically active isoforms of VEGF. In collaboration with Prof. Gera Neufeld (from the Technion’s Department of Biology) we have characterized the biological activity of the various isoforms and the receptors mediating VEGF signaling.
In collaboration with Dr. Jacob Pitcovski from Migal LTD, we have used the baculovirus system for the expression and partial purification of infectious bursal disease virus (IBDV) coat protein VP2 and VP3 demonstrated that VP2 confers full protection in chicks against infectious bursal disease virus.
2) The study of the baculovirus based expression system. We have characterized the promoter region of the polyhedrin promoter used in the baculovirus expression system and showed that a host encoded DNA-binding protein acts as a negative regulator and is involved in the delayed expression of polyhedrin promoter. The results support the concept of negative regulation. Thus, our studies indlicated that baculovirus utilizes an insect cell derived nuclear factor to control the differential expression of late versus very late genes.
In addition, we have constructed a bi-cistronic expression vectors for the baculovirus expression system.

Levi,, B., Z., and Ozato K. 1990. The use of the baculovirus expression system for the study of DNA binding proteins. In White, M., and Reuveny, S. (Eds.). Biologicals from recombinant microorganisms and animal cells-New strategies in production recovery. VCH (Verlag Chemie, F.R. Germany) and Balaban Publisher (Rehovot, Israel).

Neutra, R., Levi, B.Z., Shoham, Y. Optimization of protein-production by the baculovirus expression system in shake flasks. Appl. Microbiol. Biotechnol. 37: 74-78,1992.

Cohen, T., Gitay-Goren, H., Neufeld, G., Levi, B.Z. High levels of biologically active vascular endothelial growth factor are produced by the baculovirus expression system. Growth Factors 7: 131-138, 1992.

Etkin, E., Carp, L., Levi, B.-Z. Spodoptera frugiperda sf-9 cells nuclear factor binds to a specific sequence on the baculovirus polyhedrin promoter. Virus Res. 31:343-356, 1994.

Pitcovski, J., Di-Castro, D., Shaaltiel, A., Azriel, A., Gutter, B., Yarkoni, E., Michael, A., Krispel, S., Levi, B.Z. Insect-cell derived VP2 of infectious bursal disease virus confers protection against the disease in chickens. Avian Diseases 40:753-761, 1996

Pitcovski, J., Goldberg, D., Levi, B. Z., Di-Castro, D., Azriel, A., Krispel, S., Maray, T., and Shaaltiel, Y. Coding region of segment A sequence of a very virulent isolate of IBDV- comparison with isolates from different countries and virulence. Avian. Dis. 42:497-506, 1998.

Pitcovski J, Levi BZ, Maray T, Di-Castro D, Safadi A, Krispel S, Azriel A, Gutter B, Michael A: Failure of viral protein 3 of infectious bursal disease virus produced in prokaryotic and eukaryotic expression systems to protect chickens against the disease. Avian.Dis. 43:8-15, 1999.

Finkelstein Y, Faktor O, Elroy-Stein O, Levi B-Z. The use of bi-cistronic transfer vector for the baculovirus expression system. J. Biotechnol. 75:33-44, 1999.

Molecular and biochemical techniques are used for the overexpression and purification of targted enzymes

Neutra R., B-Z. Levi and Y. Shoham. Optimization of protein-production by the baculovirus expression vector system in shake flasks. Appl. Microbiol. Biotechnol. 37:74-78 1992.

Lundgren, K. R., L. Bergkvist, S. Hogman, H. Joves, G. Eriksson, T. Bartfai, J. van der Laan, E. Rosenberg and Y. Shoham. TCF mill trial on softwood pulp with Korsnas thermostable and alkaline stable xylanase T-6. FEMS Microbiol. Rev. 13:365-368, 1994.

Fishman A., Z. Berk, and Y. Shoham. Large scale purification of xylanase T-6. Appl. Microbiol. Biotechnol 44: 88-93, 1995.

Lapidot, A., A. Mechaly, and Y. Shoham. Overexpression and single step purification of a thermostable xylanase from Bacillus stearothermophilus T-6. J. Biotechnol. 51:259-264, 1996.

Mechaly, A., A. Teplitsky, V. Belakhov, T. Baasov, G. Shoham and Y. Shoham. Overproduction and characterization of seleno-methionine xylanase T-6. J. Biotechnol. 78:83-86, 2000