DEPT OF BOTANY
| Researcher : Chen SF |
| Project Title: | Metabolic engineering for enhanced astaxanthin biosynthesis in Chlorella zofingiensis (Chlorophyta) |
| Investigator(s): | Chen SF, Huang J |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2007 |
| Completion Date: | 12/2009 |
| Abstract: |
| To establish a chlorella transformation system; to introduce a heterologous carotenoid hydroxylase gene into C. zofingiensis; to study the regulation of astaxanthin biosynthesis in the engineered host; to maximize the production of astaxanthin in the genetically manipulated C. zofingiensis. |
| Project Title: | Genetic enineering of a green algal phytoene desaturase for herbicide resistance and its use as a selectable marker for nuclear transformation |
| Investigator(s): | Chen SF, Huang J |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 03/2008 |
| Completion Date: | 09/2009 |
| Abstract: |
| The eukaryotic green algae are photoautotrophs, requiring only sunlight, carbon dioxide, water and basic nutrients for maximal growth. Because they potentially provide all the benefits of higher plants with large-scale microbiol production approaches, green microalgae may be an attractive alternative to the other microbial systems currently in use. Microalgae have already served as a major natural source of valuable molecules including carotenoids, long-chain polyunsaturated fatty acids and phycocolloids. In recent years, there has been a surge of interest in microalgal metabolites as a source of novel molecules. However, it is difficult to produce stable transformants of most microalgae due to the lack of proper selectable markers and promoters to drive heterologous gene expression. Chlamydomonas reinhardtii is a widly used model system for basic biological and ecological studies, especially those that cannot be investigated in bacteria and yeast (Rochaix, 1995). The recently developed techniques for nuclear and organelle transformation (Boynton et al., 1988; 1993; Kindle, 1990) have greatly expanded the use of this alga as a model system. However, it is difficult for C. reinhardtii to express heterologous genes. Therefore, the development of dominant selectable markers is still severely hampered. Efficient transformation systems for algae commonly rely on homologous genes that complement auxotrophic mutations which are unavailable for most algae. For the expression of heterologous genes, homologous promotor and 3' regions providing a cognate polyadenylation signal are highly required. Up to date, only a few selectable markers are available for a few model organisms, such as the green alga C. reinhardtii, the diatom Phaeodactylum tricornutum, etc. Compared with higher plants, the transformation of algae is still in its infancy because no unviversal tools (e.g. expression cassette and selectable markers) as those used in higher plants (e.g. 35S promoter and Agrobacterium transformation system) are available for algae. Carotenoids are essential components of the photosynthetic apparatus. They participate in light harvesting and protect the chloroplasts from the harmful effect of single oxygen formed during photosynthesis (Windhovel et al., 1994). The enzyme phytoene desaturase (PDS) is the main target for herbicides that prevent the formation of zeta-carotene, resulting in the degradation of chlorophyll and the destruction of chloroplast membranes. Mutations (some amino acid substitutions) of the cyanobacterium Synechococcus PDS, the aquatic weed Hydrilla verticillata PDS resulted in herbicede-resistant PDS enzymes (Chamovitz et al., 1991; Michel et al., 2004), and have conferred herbicide resistance when expressed in plants (Wagner et al., 2002; Arias et al., 2006). We hypothesize that similar amino acid substitutions in PDS of eukaryotic algae could also resist to herbicide. Thus, the PDS from microalgae can be modified as dominant selectable markers used for genetic engineering of biotechnologically important microalgae for which no successful genetic manipulation is available up to date. The purpose of the proposed project is to develope a dominant selectable marker for nuclear transformantion of microalgae by modifying a homologous PDS gene that will confer herbicide resistance when expressed in algae. To achieve this project aim, the gene coding for the carotenoid biosynthetic enzyme PDS from C. reinhardtii will be isolated. This gene will be mutated and functionally analysed in recombinant E. coli and in vitro assay for herbicide resistance. Herbicide-resistant PDSs will be inserted into C. reinhardtii expressing cassete (Stevens et al. 1996; Cerutti et al., 1997; Schroda et al., 2000) and introduced into the algal cells using the method introduced by Kindle (1990). Herbicide resistance and pigment profiles of transformants will be assessed. References 1. Arias , R.S., Dayan, F.E., Michel, A., Howll, J., and Scheffler B.E. (2006) Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker. Plant Biotechnology Journal 4, 263-273. 2. Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler J.P. Johnso, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D. Klein., T.M., Shark, K.B., and Sanford, J.C. (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240, 1534-1538. 3. Cerutti, H., Johnson, A.M., Gillham, N.W. and Boynton, J.E. (1997) A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics, 145, 97-110. 4. Chamovitz, D., G. Sandmann, and J. Hirschberg. (1993) Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting stepin carotenoid biosynthesis. J. Biol. Chem. 268, 17348-17353. 5. Kindle, K.L. (1990) High-fregquency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 87, 1228-1232. 6. Rochaix, J.-D. (1995) Chlamydomonas reinhardtii as the photosynthetic yeast. Annu. Rev. Genet. 29, 209-230. 7. Schroda, M., Blocker, D., and Beck, C.F. (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J. 21, 121-131. 8. Stevens, D.R., Rochaix, J.-D. and Purton, S. (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol. Gen. Genet. 251, 23-30. 9. Wagner T., Windhovel, U., and Romer. S. (2002) Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase confers resistance to bleeching herbicides. Z Naturforsch 57, 671-679. 10. Windhovel, U., Geiges, B., Sandmann, G., and Boger, P. (1994) Expression of Erwina Uredovora phytoene desaturase in Synechococcus PCC 7942 leading to resistance against bleaching herbicides. Plant Physiol. 102, 119-125. |
| Project Title: | Directed evolution of carotenoid ketolase and hydroxylase for investigating the molecular basis of enzyme function and astaxanthin biosynthesis |
| Investigator(s): | Chen SF, Huang J |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 09/2008 |
| Abstract: |
| (1) to investigate the BKT activity toward zeaxanthin for astaxanthin production; (2) to investigate the CHYb activity toward beta-carotene for zeaxanthin production; (3) to study the molecular basis of the enzymes for activity and substrate specificity; (4) to elucidate the evolution of astaxanthin biosynthesis. |
| Project Title: | Chloroplast genome engineering of Chlamydomonas reinhardtii for metabolic pathway study |
| Investigator(s): | Chen SF, Huang J |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 05/2009 |
| Abstract: |
| Astaxanthin is a red ketocarotenoid that occurs only in a limited number of bacteria, fungi, and in certain unicellular algae (Boussiba, 2000). This red pigment has been widely used as a feed supplement in aquaculture and poultry industries (Johnson and Schroeder 1995). In addition, due to its high antioxidant activity, astaxanthin has also been used commercially as nutraceuticals and for cosmetic and pharmaceutical purposes (Lorenz and Cysewski 2000, Guerin et al. 2003). The unicellular green microalga Haematococcus pluvialis reveals the highest astaxanthin accumulation (up to 4% of dry biomass) (Boussiba et al., 1992). H. pluvialis has served as a natural source of astaxanthin. The slow growth rate and lack of a genetic transformation system of this alga, however, limit its application. In contrast, other astaxanthin-producing microorganisms have much lower cellular astaxanthin content. With very few exceptions, higher plants normally do not synthesize astaxanthin. In recent years, the carotenoid biosynthetic pathway in some plants has been extended to astaxanthin by nuclear transformation of a heterologous β-carotene ketolase (bkt) gene. However, in all cases, only trace amounts of astaxanthin were synthesized in the transgenic plants. Knowledge of ketocarotenoid biosynthesis is very limited, which greatly hinders the exploitation and application of astaxanthin-producing organisms and transgenic plants as natural sources of ketocarotenoids on an industrial scale. Recently, biosynthesis of astaxanthin in tobacco leaves was achieved by plastid transformation, which highlights the utility of plastid transformation as an excellent tool to drastically alter carotenoid compositions and contents in algae and crop plants. (Husunuma et al., 2008). The unicellular geen alga Chlamydomonas reinhardtii is an important model organism for studies of photosynthesis and pigment biosynthesis (Harris 1989). No astaxanthin has been reported to occur in C. reinhardtii although its genome contains an open reading frame with strong similarity to BKT enzymes from the green algae H. pluvialis and Chlorella zofingiensis (Grossman et al. 2004, Huang et al. 2006a, b). We have recently characterized the putative Chlamydomonas BKT and found it to possess ketolase activity. RT-PCR detection revealed that the expression of the bkt gene was too low to trigger the cell to produce astaxanthin. We therefore hypothesized that the low expression of the bkt gene is the limiting factor for C. reinhardtii to synthesize ketocarotenoids. To prove the hypothesis, we introduced a Haematococcus bkt into C. reinhardtii by nuclear transformation. However the genetic modified cells failed to produce significant amounts of ketocarotenoids even though all the elements required for optimal transcription and translation (promoter, intron and other regulatory regions) had been included in the chimeric gene construction. RT-PCR detection revealed very low expression of the transgene, possibly resulting from the so-called gene silencing. Very recently, robust expression of heterologous genes has been achieved in Chlamydomonas chloroplast by plastid transformation (Mayfield et al., 2007). Transgene expression from the chloroplast genome offers several advantages over nuclear transformation, including high-level accumulation of foreign proteins, transgene stacking in operons and a lack of epigenetic interference with the stability of transgene expression (Bock 2007). Thus we propose to solve the problem of low expression of transgenes in C. reinhardtii by chloroplast genome engineering. The purpose of this proposed research is to develop a model system for the study of astaxanthin biosynthesis in green algae. The unicellular green alga C. reinhardtii, to which its chloroplast DNA can be easily transformed, will serve as a target organism and genetically be engineered by introducing a novel copy of bkt gene into its chloroplast via plastid transformation. It is expected that the modified cells will produce and accumulate astaxanthin in the chloroplast which takes up about half of the cell volume. The established system will help to reveal the regulation of astaxanthin biosynthesis in the green alga H. pluvialis, the organism with the richest astaxanthin content in nature. References Bock R. (2007) Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming. Curr. Opin. Biotechn. 18: 100-106. Boussiba (2000) Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol. Plantarum. 108:111-117. Boussiba, S., Fan, L. & Vonshak, A. 1992. Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. Method Enzymol. 213: 386-391. Grossman, A. R., Lohr, M. & Im, C. S. 2004. Chlamydomonas reinhardtii in the landscape of pigments. Annu. Rev. Genet. 38:119-173. Guerin, M., Huntley, M. E., & Olaizola, M. 2003. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol. 21:210-216. Harris, E. H. The Chlamydomonas Sourcebook, Academic Press, San Diego, 1989 Huang, J.C., Wang, Y., Sandmann, G. & Chen, F. 2006a. Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta). Appl. Microbiol. Biotechnol. 71:473-9. Huang, J.C., Chen, F. & Sandmann, G. 2006b. Stress-related differential expression of multiple beta-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis. J. Biotechnol. 122:176-85. Johnson, E. A. & Schroeder, W. A. 1995. Microbial carotenoids. Adv. Biochem. Eng. Biotechnol. 53:119-178. Lorenz, R. T. & Cysewski, G. R. 2000. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol. 18:160-167. Mayfield S.P., Manuell A.L, Chen S. et al. (2007) Chlamydomonas reinhardtii chloroplasts as protein factories. Curr. Opin. Biotechn. 18:126-133. |
| Project Title: | Assess the potential of the green microalgae Chlorella species as biodiesel feedstocks |
| Investigator(s): | Chen SF, Huang J |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 03/2010 |
| Abstract: |
| Petroleum fuels are recognized as unsustainable due to their depleting supplies and contribution to global warming (Chisti, 2008). Renewable biofuels are promising alternatives to petroleum fuels, among which biodiesel has attracted the most attention in recent years (Knothe et al., 1997, 2006; Fukuda et al., 2001; Gerpen, 2005; Meher et al., 2006; Hu et al., 2008). Biodiesel is a diesel-equivalent fuel derived from biological feedstocks and is chemically referred to as a fatty acid methyl ester (FAME). In comparison with traditional fuels, biodiesel is carbon neutral, contributes less emission of gaseous pollutants and so is environmentally beneficial (Ma and Hanna, 1999). Current biodiesel is mainly derived from vegetable oils, animal fats and waste cooking oils (Knothe et al., 1997; Lang et al., 2001; Al-Widyan et al., 2002; Zhang et al., 2003; Demirbas, 2005). However, such sources of biodiesel cannot meet the existing need for transport fuels as immense cultivation lands have to be taken up for oil crops (Chisti, 2007). The relatively high cost of using vegetable oil as feedstock also restricts the commercialization of biodiesel (Lang et al., 2001). Microalgae are sunlight-driven cell factories that convert carbon dioxide to potential biofuels, foods, feeds and high-value bioactives (Spolaore et al., 2006). Microalgae have being considered as feedstocks for biodiesel production because of their rapid growth and high contents of lipids (Chisti, 2007; Hu et al., 2008). Unlike oil crops, microalgae can be easily cultured in outdoor ponds or bioreactors (Chen, 1996; Del Campo et al., 2007; Pruvost et al., 2009), making it be a more competent alternative to oil crops in biomass production. When consider a microalgal feasibility for mass biodiesel production, two key factors, namely cell biomass and lipid content are essential for the initial assessment. Thus microalgae with fast-growth and high lipid productivity are desirable for biodiesel exploitation. The green microalgae Chlorella species consist of about 10 species, most of which can grow fast photoautotrophically, mixotrophically and heterotrophically (Chen, 1997; Shi et al., 2002; Ip et al., 2004; Miao and Wu, 2004). High contents of lipids have been reported in some of the species, such as C. vulgaris and C. protothecoides (Miao and Wu, 2004; Liu et al., 2008; Hsieh and Wu, 2009). Compared with photoautotrophic culture, heterotrophic culture can obtain much higher biomass as demonstrated by C. zofingiensis that at least 5-fold biomass was obtained by heterotrophically culturing the alga using glucose as sole carbon and energy source (Sun et al., 2008). The heterotrophic ability of Chlorella species also eliminates light requirement that is essential for photoautotrophic microalgae and therefore reduces the cost in the production process (Borowitzka, 1999). The purpose of this proposed project is to assess several Chlorella species for their potential as novel sources of biodiesel by the investigation of their heterotrophic growth and fatty acid productivity with various organic carbon sources and different stress conditions. References Al-Widyan, M.I., Al-Shyoukh, A.O., 2002. Experimental evaluation of the transesterification of waste palm oil into biodiesel. Bioresour. Technol. 85, 253-256. Borowitzka, M.A., 1999. Commercial production of microalgae: ponds, tanks, tubes and fermenters. J. Biotechnol. 70, 313-321. Chen, F., 1996. High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol. 14, 421-426. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126-31. Del Campo, J.A., Garcia-Gonzalez, M., Guerrero, M.G., 2007. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl. Microbiol. Biotechnol. 74, 1163-1174. Demirbas, A., 2005. Biodiesel production from vegetable oils via catalytic and non-catalytic supercritical methanol transesterification methods. Prog. Energy. Combust. Sci. 31, 466-487. Fukuda, H., Kondo, A., Noda, H., 2001. Biodiesel fuel production by transesterification of oils. J. Biosci. Bioeng. 92, 405-416. Gerpen, J.V., 2005. Biodiesel processing and production. Fuel Process. Technol. 86, 1097-1107. Hsieh, C.-H., Wu, W.-T., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100, 3921-3926. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621-639. Ip, P.F., Wong, K.H., Chen, F., 2004. Enhanced production of astaxanthin by the green microalga Chlorella zofingiensis in mixotrophic culture. Process Biochem. 39, 1761-1766. Knothe, G., 2005. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process. Technol. 86, 1059-1070. Knothe, G., Dunn, R.O., Bagby, M.O., 1997. Biodiesel: the use of vegetable oils and their derivatives as alternative diesel fuels. in: B.C. Saha, J. Woodward (Eds.), Fuels and Chemicals from Biomass. American Chemical Society, Washington, D.C., pp. 172-208. Lang, X., Dalai, A.K., Bakhshi, N.N., Reaney, M.J., Hertz, P.B., 2001. Preparation and characterization of bio-diesels from various bio-oils. Bioresour. Technol. 80, 53-62. Liu, Z.-Y., Wang, G.-C., Zhou, B.-C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99, 4717-4722. Ma, F., Hanna, M.A., 1999. Biodiesel production: a review. Bioresour. Technol. 70, 1-15. Meher, L.C., Vidya Sagar, D., Naik, S.N., 2006. Technical aspects of biodiesel production by transesterification--a review. Renew. Sustain. Energy Rev. 10, 248-268. Miao, X., Wu, Q., 2004. High yield bio-oil production from fast pyrolysis by metabolic controlling of Chlorella protothecoides. J. Biotechnol. 110, 85-93. Miao, X., Wu, Q., 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97, 841-846. Pruvost, J., Van Vooren, G., Cogne, G., Legrand, J., 2009. Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor. Bioresour. Technol. 100, 5988-5995. Shi, X.-M., Chen, F., 2002. High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture. Biotechnol. Prog. 18, 723-727. Sun, N., Wang, Y., Li, Y.-T., Huang, J.-C., Chen, F., 2008. Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta). Process Biochem. 43, 1288-1292. Zhang, Y., Dub, M.A., McLean, D.D., Kates, M., 2003. Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresour. Technol. |
| Researcher : Chye ML |
| Project Title: | Investigations on stress-inducible Arabidopsis acyl-CoA binding proteins |
| Investigator(s): | Chye ML |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 09/2007 |
| Abstract: |
| To select a lead-binding ACBP and to verify feasibility of its application in phytoremediation; to study Arabidopsis ACBP3 in response to phytopathogen stress and to verify its use in protection against phytopathogens. |
| Project Title: | Outstanding Researcher Award 2006-2007 |
| Investigator(s): | Chye ML |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Outstanding Researcher Award |
| Start Date: | 11/2007 |
| Abstract: |
| Nil |
| Project Title: | Characterization of a cDNA encoding a 71-kD rice acyl-CoA-binding protein |
| Investigator(s): | Chye ML |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2009 |
| Completion Date: | 03/2010 |
| Abstract: |
| Purpose: Acyl-CoA-binding proteins (ACBPs) show conservation in the acyl-CoA-binding domain and thus they can bind and transport acyl-CoA esters in lipid metabolism. The aim of this project is to characterize a potentially unusual member belonging to the family of ACBPs in the monocot, rice (Oryza sativa). This particular rice ACBP, designated OsACBP1, has a predicted chloroplast targeting sequence and two potential transmembrane domains. At present, no chloroplast targeting sequence has been identified in known plant ACBPs. In plants, de novo fatty acid biosynthesis occurs in the chloroplast and acyl-CoAs (oleoyl-CoA and palmitoyl-CoA) esters are subsequently transported via the cytosol to the endoplasmic reticulum (ER) for non-plastidial lipid metabolism. Presence of a chloroplast form of ACBP would indicate that OsACBP1 may function in acyl-CoA trafficking within the chloroplast and suggests a new role for ACBPs in this subcellular compartment. In the dicot model plant, Arabidopsis, we have characterized a family of six ACBPs but none of these include a chloroplastic form. ACBP4 and ACBP5 are candidates that mediate the cytosolic transfer of oleoyl-CoA esters originating from the chloroplast to the ER, based on our observations that recombinant His-tagged ACBP4 and ACBP5 bind oleoyl-CoA esters in vitro. In this project we will characterize the putative chloroplastic form of ACBP from rice. We will test if the predicted chloroplast targeting sequence in OsACBP1 functions in directing it to the chloroplast by using a Green Fluorescent Protein (GFP) autofluorescence tag in transient expression assays on subcellular localization. Further, we will identify the acyl-CoA esters that bind OsACBP1 in in vitro binding assays using recombinant His-tagged OsACBP1 expressed in E. coli. A His-tag will be fused to OsACBP1 to facilitate easy purification of the recombinant protein in mass production for in vitro assays with oleoyl-CoA and palmitoyl-CoA. Key issues: In Arabidopsis, six ACBPs have been identified and characterized (Leung et al., 2004). They, designated ACBP1 to ACBP6, have been subcellularly localised to various compartments but none is targeted to the chloroplast. ACBP1 and ACBP2 are localized in the plasma membrane (Chye et al., 1999; Chye et al., 2000; Li and Chye, 2003; 2004), ACBP3 is extracellularly-targeted (Leung et al., 2006) and ACBP4, ACBP5 and ACBP6 are cytosolic proteins (Leung et al., 2004; Chen et al., 2008). These ACBPs differ in molecular mass, ranging from 10 to 73 kDa and substrate specificities for acyl-CoA esters (Leung et al., 2004; Gao et al., 2008). We have demonstrated that the genes encoding Arabidopsis ACBP1, ACBP2, ACBP3, and ACBP6, are subject to regulation by various forms of abiotic and biotic stress (Chen et al, 2008; Gao et al, 2008; Li et al., 2008; Xiao et al, 2008a, 2008b). The overexpression of ACBP1 or ACBP2 in transgenic Arabidopsis resulted in tolerance to heavy metal stress and oxidative stress because ACBP1 and ACBP2 are associated with membrane repair (Xiao et al, 2008a; Gao et al, 2008). ACBP3-overexpressing transgenic Arabidopsis show enhanced resistance to pathogens while ACBP6-overexpressing transgenic Arabidopsis are conferred freezing tolerance (Chen et al, 2008). ACBPs mediate lipid transfer and the manipulation of lipid composition has resulted in stress-tolerance. These initial investigations on the model plant, Arabidopsis, have many advantages including the availability of its complete genome sequence and knock-out mutants. Further, Arabidopsis has a rapid life-cycle, is easy to transform and maintain in the laboratory. If we were to carry out similar investigations on rice, it would have taken us a longer time to achieve similar results in understanding the role of plant ACBPs because rice is difficult to transform and knock-out mutants are not easily accessible. Our eventual goal is to genetically-modify rice to stress-resistance by manipulation of the rice genome using Arabidopsis ACBPs. But before we can overexpress Arabidopsis ACBPs in transgenic rice to obtain these stress-resistant varieties by application of the principles used in the generation of stress-tolerant transgenic Arabidopsis, we should first attempt to acquire basic knowledge on the endogenous ACBP family in rice. From GenBank data, we have identified six rice genes that encode proteins conserved in the acyl-CoA binding domain. Of these, three resemble Arabidopsis ACBP6, because they consist only of an acyl-CoA-binding domain. Another has ankyrin repeats and a membrane domain, resembling Arabidopsis ACBP1 and ACBP2. However, the largest rice ACBP (71-kD), designated OsACBP1, possesses an unusual predicted structure in comparison to all known plant ACBPs. Computer predictions suggest that OsACBP1 contains kelch motifs, (resembling ACBP4 and ACBP5), as well as two transmembrane domains and an N-terminal chloroplast targeting sequence. The presence of two predicted transmembrane domains and a potential chloroplast targeting sequence is novel to plant ACBPs. There have been no reports of any ACBP subcellularly localized in the chloroplast. Problems addressed: The problems addressed in this project are: 1. to establish if the predicted chloroplast targeting sequence in OsACBP1 is functional; 2. to identify the acyl-CoA esters that bind OsACBP1. Although fatty acid biosynthesis initiates from within the chloroplast, none of our previously characterized Arabidopsis ACBPs have been localized to this subcellular compartment. Since rice is a monocot, unlike Arabidopsis, it may possibly have evolved a chloroplastic form of ACBP to facilitate an efficient transfer of acyl-CoA esters within the chloroplast to cytosolic ACBPs. To establish if the predicted chloroplast targeting sequence of OsACBP1 functions in targeting to the chloroplast, its N-terminal predicted targeting sequence consisting of the first 30 amino acids will be fused in-frame to autofluorescent protein GFP. Also, the full-length OsACBP1 peptide will be fused in-frame to GFP as comparison. The acyl-CoA substrates that bind OsACBP1 will be identified using in vitro binding assays. Oleoyl-CoA and palmitoyl-CoA will be tested because they are generated from de novo fatty acid biosynthesis inside chloroplasts. References: 1. QF Chen, S Xiao and ML Chye. 2008. Plant Physiology 148: 304-315. 2. ML Chye, BQ Huang and SY Zee. 1999. Plant Journal 18: 205-214. 3. ML Chye, HY Li and MH Yung. 2000. Plant Mol Biol: 44: 711-721. 4. W Gao, S Xiao, HY Li and ML Chye. 2008. New Phytologist doi: 10.1111/j.1469-8137.2008.02631.x 5. KC Leung, HY Li, G Mishra and ML Chye. 2004. Plant Mol Biol 55: 297-309. 6. KC Leung, HY Li, S Xiao, MH Tse, ML Chye. 2006. Planta 223: 871-881. 7. HY Li and ML Chye. 2003. Plant Mol Biol 51: 483-492. 8. HY Li and ML Chye. 2004. Plant Mol Biol 54: 233-243. 9. HY Li, S Xiao and ML Chye. 2008. J. Exp. Bot. 59: 3997-4006. 10. S Xiao, W Gao, QF Chen, S Ramalingam and ML Chye. 2008a. Plant Journal 54: 141-151. 11. S Xiao, HY Li, JP Zhang, SW Chan and ML Chye. 2008b. Plant Mol. Biol. 68: 571-583. |
| Project Title: | The Third Asian Symposium on Plant Lipids (ASPL 2009) ARABIDOPSIS ACYL-COENZYME-A BINDING PROTEIN 2 INTERACTS WITH A LYSOPHOSPHOLIPASE |
| Investigator(s): | Chye ML |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 11/2009 |
| Completion Date: | 11/2009 |
| Abstract: |
| N/A |
| Project Title: | Analysis of transgenic model plants |
| Investigator(s): | Chye ML |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2010 |
| Abstract: |
| Purpose: Plant isoprenoids include natural compounds that promote physiological functions and defense against phytopathogens. 3-Hydroxy-3-methyl-glutaryl-CoA synthase (HMGS) is an enzyme in isoprenoid biosynthesis in eukaryotes and some bacteria. The aim of this project is to address whether isoprenoid production can be increased in transgenic plants by the overexpression of wild-type/mutant HMGS. It will also show whether physiological functions (e.g., plant growth and development) and defense against phytopathogens are consequently enhanced in HMGS-overexpressors. Key issues: Isoprenoids are one of the largest classes of natural products including sterols, sesquiterpenes, polyterpenes, carotenoids, taxol and artemisinin, and are mainly produced by plants (Bach, 1995; Roberts, 2007). Plant isoprenoids are essential for membrane biogenesis (sterols), photosynthesis (carotenoids, chlorophyll), respiration (quinones), growth and development (abscisic acid and gibberellic acid) and in disease resistance (sesquiterpenes, brassinosteroids), and they also play significant roles in plant-environment interactions, plant-plant communication, and plant-insect interactions (Bach, 1995; Pichersky and Gershenzon, 2002). Isoprenoids such as natural rubber are economically valuable while monoterpenes (e.g., linalool) are used as flavors/fragrances in food additives. Taxol and artemisinin are important anti-cancer drugs while artemisinin is anti-malarial too. The important functions of many isoprenoids make it useful for them to be overproduced in transgenic plants. Two biosynthetic pathways synthesize isoprenoid precursors in plants, the cytosolic mevalonate (MVA) pathway (Bach 1995) and the plastid-confined non-MVA pathway (Lichtenthaler 1999; Rohmer 1999). The MVA pathway provides isoprenoids essential for growth, development and defense (Bach, 1995; Hemmerlin and Bach 1998; Hemmerlin et al. 1999). Both HMGS and HMG reductase (HMGR, Kush et al., 1990; Chye et al., 1992) catalyze the early steps in the MVA pathway. HMGS catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield HMG-CoA and HS-CoA while HMGR converts HMG-CoA to MVA. My laboratory has cloned and characterized cDNAs encoding four isoforms of Brassica juncea HMGS, designated BjHMGS1 to BjHMGS4 (Alex et al., 2000; Nagegowda et al., 2004; 2005). Brassica HMGS expression is stress-inducible and is highly expressed during early development in flower, seed and seedling, indicative of its role in development (Alex et al., 1999). Although BjHMGS1 to BjHMGS4 are highly-conserved, they are differentially expressed during floral and seedling development (Nagegowda et al., 2005). BjHMGS1 was localized in the cytosol by confocal microscopy and western blot analysis of subcellular protein fractions from transgenic plants expressing Green Fluorescent Protein-tagged BjHMGS1 (Nagegowda et al., 2005). The structure of BjHMGS1 in molecular interaction with its inhibitor F-244 has been elucidated to explore the potential of using HMGS as an alternative target for anti-cholesterol and antibiotic drugs, given that MVA is a precursor of cholesterol in mammals and HMGS inhibition adversely affects bacterial viability (Pojer et al., 2006). We first report on the biochemical characterization of a plant-derived HMGS using bacterial-expressed recombinant (His)6-tagged BjHMGS1 (Nagegowda et al., 2004). Catalytic residues in BjHMGS1 were identified by site-directed mutagenesis (Nagegowda et al., 2004). (His)6-BjHMGS1 was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased Vmax, indicating some involvement of these residues in catalytic capacity. Unlike (His)6-BjHMGS1, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Subsequently, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that H188 is involved in conferring substrate inhibition to (His)6-BjHMGS1 (Nagegowda et al., 2004). The identification of BjHMGS1 mutants which are substrate insensitive and show enhanced enzymatic activity (Nagegowda et al., 2004) provides us the tools for the metabolic engineering of isoprenoids. Investigations on the overexpression of these mutant BjHMGS1 in model plants is a first step towards this end. Specifically, this project will investigate the effects in the overexpression of wild-type and mutant (H188N, S359A and H188N/S359A) BjHMGS1 in model plants, Arabidopsis and tobacco. The following problems will be addressed: 1. Does the overexpression of wild-type or mutant BjHMGS1 improve isoprenoid accumulation in model plants and if it does, what is the identity of the isoprenoid compounds that accumulate? 2. Which version (wild-type/mutant) of BjHMGS1 is most effective in causing isoprenoid accumulation when overexpressed in transgenic plants? 3. Does the overexpression of wild-type/mutant BjHMGS1 affect plant growth, development and defense against phytopathogens? To examine isoprenoid accumulation, isoprenoid products such as the various plant sterols (campesterol, sitosterol and stigmasterol) and sesquiterpenes will be measured in transgenic Arabidopsis and tobacco lines. Plant sterols will be measured because they are useful as functional foods as they can block intestinal cholesterol adsorption and lower cholesterol in humans (Ostlund et al., 2003). Sitosterol further promotes plant growth and development while sesquiterpenes enhance plant defense. Transgenic Arabidopsis and tobacco will also be examined for changes in growth using germinating seedlings, and for any improved disease resistance to Botrytis cinerea (fungal pathogen). These proposed investigations using model plants are prerequisite to the application of wild-type/mutant BjHMGS1 in transgenic Artemisia which produces artemisinin (a sesquiterpene) via the MVA pathway or in crops for improved growth and protection against phytopathogens, as well as for the production of sterol-enriched plant products in functional foods to control cholesterol adsoprtion. References: 5-year IF(2008):PNAS 10.228;Plant J 6.951;Plant Physiol 6.650;Biochem J 4.251;Plant Mol Biol 3.901;Planta 3.304 1. Alex D, Bach TJ, Chye ML (2000) Plant J 22:415-426. 2. Bach TJ (1995) Lipids 30:191-202. 3. Chye ML, Tan CT, Chua NH (1992) Plant Mol Biol 19: 473-484. 4. Dhawan R et al. (2009) Plant Cell 21:1000–1019. 5. Ferrari S et al. (2003) Plant J 35:193-205. 6. Hemmerlin A, Bach TJ (1998) Plant J 14:65-74. 7. Hemmerlin A et al., (1999) Acta Bot Gall 146:85-100 8. Kush A, Goyvaerts E, Chye ML, Chua NH (1990) Proc Natl Acad Sci USA 87:1787-1790. 9. Lichtenthaler HK (1999) Annu Rev Plant Physiol Plant Mol Biol 50:47-65. 10. Nagegowda DA, Bach TJ, Chye ML (2004) Biochem J 383:517-527. 11. Nagegowda DA, Ramalingam SK, Hemmerlin A, Bach TJ, Chye ML (2005) Planta 221:844-856. 12. Ostund RE et al. (2003) American J Clin Nutr 77:1385-1389. 13. Pichersky E, Gershenzon J (2002) Curr. Opin. Plant Biol. 5: 237-243. 14. Pojer F, Ferrer JL, Richard SB, Nagegowda DA, Chye ML, Bach TJ, Noel JP (2006) Proc Natl Acad Sci USA 103:11491-11496. 15. Roberts SC (2007) Nature Chemical Biology 3: 387-395. 16. Rohmer M (1999) Nat Prod Rep. 16:565-574. 17. Schaller H, Grausem B, Benveniste P, Chye ML, Tan CT, Song YH, Chua NH (1995) Plant Physiol 109:761- 770. |
| Researcher : Corke H |
| Project Title: | 2nd International Wheat Quality Conference Effect of Wheat Quality on Noodle Production |
| Investigator(s): | Corke H |
| Department: | Botany |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 05/2001 |
| Abstract: |
| N/A |
| Project Title: | Molecular markers for starch content and quality in rice |
| Investigator(s): | Corke H |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 11/2006 |
| Completion Date: | 10/2009 |
| Abstract: |
| To develop gene-tagged markers and their integration into a genetic linkage map, and mapping OTL for starch content and quality (structural and functional properties); to sequence major genes, analysis of gene diversity and linkage disequilibrium, and association mapping of the genes in relation to starch content and quality; to develop a protocol for marker-assisted selection to most effectively simulate multiple components contributing to high starch contents and desired starch properties in Chinese rice breeding and therefore with direct applicability to economic development in China. |
| Project Title: | ASA-CSSA-SSSA International Annual Meetings 2009 Molecular markers for starch quality of rice |
| Investigator(s): | Corke H |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 11/2009 |
| Completion Date: | 11/2009 |
| Abstract: |
| N/A |
| Researcher : Li H |
| List of Research Outputs |
| Xu X., Li X.Y., Li X.Z. and Li H., Degradation of melatonin by UV, UV/H2O2, Fe2+/H2O2 and UV/Fe2+/H2O2 processes, Separation and Purification Technology. 2009, 68: 261-266. |
| Researcher : Ma CY |
| Project Title: | Analysis of chemically modified non-starch polysaccharides by Raman spectroscopy |
| Investigator(s): | Ma CY |
| Department: | Botany |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 11/2003 |
| Abstract: |
| To apply the technique of Raman spectroscopy for determining degrees of chemical modification in proteins, and extend the technique to study modified non-starch polysaccharides. |
| Project Title: | Institute of Food Technologists Annual Conference 2009 Conformational study of SPI treated with ultrasound |
| Investigator(s): | Ma CY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 06/2009 |
| Abstract: |
| N/A |
| Project Title: | 15th European Carbohydrate Symposium (eurocarb 15) Raman and FTIR spectroscopic study of sulfated non-starch polysaccharides |
| Investigator(s): | Ma CY |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 07/2009 |
| Completion Date: | 07/2009 |
| Abstract: |
| N/A |
| Researcher : Tsang JSH |
| Project Title: | Molecular characterization of a novel bacterial permease that transport haloacid |
| Investigator(s): | Tsang JSH |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 10/2006 |
| Completion Date: | 03/2010 |
| Abstract: |
| (1) Confirmation of the expression of the putative permease gene; (2) confirmation of the putative gene as a second member of a haloacid operon; (3) functional analysis of the putative permease; (4) structural analysis of the putative permease protein. |
| Project Title: | Utilization of a microfluidic system for teaching of biotechnology, microbiology and molecular biology |
| Investigator(s): | Tsang JSH |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Run Run Shaw Research and Teaching Endowment Fund - Teaching Grants |
| Start Date: | 08/2008 |
| Abstract: |
| The beginning of the twenty-first century can be described as the decade for biological sciences. Since the year 2000 The Nobel Prizes in Medicine have been award to researchers working on many disciplines of biological systems. These include genetic regulations of programmed cell death and cell cycle control, signal transduction in the nervous system, gene silencing by means of double-stranded RNA, discovery of the bacterium Helicobacter pylori and most recently on the use of embryonic stem cells. Moreover, four of the seven Nobel Prizes in Chemistry since the beginning of the decade were also given to researchers in the biological fields. These include methods used for structural analysis of biological macromolecules, channels in cell membranes, ubiquitin-mediated protein degradation and the molecular basis of eukaryotic transcription. In order to teach students enrolled in the biological sciences programmes it is necessary to ensure that the most updated information will be delivered to these students. Today the major techniques used for analysis of biological systems involve the studies of DNA, RNA and protein. Preparative and analytical electrophoresis systems were normally used for these investigations. Traditionally, agarose and polyacrylamide gel electrophoresis systems were used for separation of these biomolecules. Since these biomolecules have different abundance and properties in the cell it is naturally necessary to enhance the detection of these substances with the addition of specific reagents. Ethidium bromide is usually used to bind to nucleic acids and help visualize the presence of DNA and RNA. In this detection method the samples in the gel were identified with the illumination of ultraviolet light. Since the visualization processes depended on the binding of the chemical to the biomolecules this reagent is unavoidably hazardous. Ethidium bromide is a chemical carcinogen and has been one of the chemical pollutants being transmitted easily without any notice. Ultraviolet light has also been used as a physical mutagen. It can also causes sever damage to the eyes when no protection was provided. The skins can also get burnt without any tanning effect when exposed to ultraviolet light. This obviously posed certain level of danger to the user and to the workers in the surrounding environment. Polyacrylamide has been used for the separation of proteins and for small sized DNA and/or RNA. In the old times people used to use their finger to check whether the acrylamide gel has been set or not. It has later been found that acrylamide is a neurotoxin. The use of these chemicals in teaching biological sciences subjects has to be exceptionally careful in order not to radiate their harmful effect. The use of a microfluidic automated electrophoresis system has the advantage of maintaining the separation functions while removing the hazardous aspect. No harmful chemical is used in the automated system and students will be able to appreciate the safety and usefulness of this kind of technology. When the traditional electrophoresis systems were used for analysis of DNA, RNA and protein, the sensitivities of these detection methods required the use of relatively large amount of materials. Samples containing DNA or RNA with less than 100 nanograms may not be readily visualized by the ethidium bromide staining method. Protein samples with less than micrograms of protein may not be visible in the Coomassie blue stained polyacrylamide gels. This will require the researcher to prepare more starting material and ends up in a much bigger volume that is difficult to handle. Moreover, due to the limitation of the detection chemicals the amounts of the biomolecules may not be quantified in these systems. The florescence of the ethidium bromide bound nucleic acids and the intensity of the Coomassie blue stained proteins do not necessarily show a linear relationship with the quantity loaded into the gel. The automated electrophoresis system uses a microfluidic technology that is much more sensitive than these traditional methods. The system is able to detect as less as nanogram of DNA, RNA and protein. This is hundred times more sensitive than the conventional method. The volume required is also minimized. This will increase the accuracy of the analysis and decrease the material that is required. Moreover, the microfluidic system makes use of highly sensitive non-harmful fluorescent dyes. This allows the abundance of the individual biomolecules to be detected and quantified. Today, we do not just talk about teaching; we will also have to talk about learning. It is necessary to provide more actual hands-on practices to the students because it can help the students to acquire and memorize the knowledge better. With the students knowing what the established methods can do, innovative and more accurate methods will be introduced for the students to compare the differences. This will train the students to learn and think more critically. In this proposal I would like to request for funding for the purchase of a microfluidic automated electrophoresis system for teaching of courses offered for the biology, biotechnology and microbiology programmes. |
| Project Title: | IUMS 2008 XII International Congress of Bacteriology and Applied Microbiology Cloning of a putative regulator of the haloacid operon of Burkholderia cepacia MBA4 |
| Investigator(s): | Tsang JSH |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 08/2008 |
| Abstract: |
| N/A |
| Project Title: | Cloning and characterization of a transcriptional regulator of the haloacid operon of Burkholderia sp. MBA4 |
| Investigator(s): | Tsang JSH |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Small Project Funding |
| Start Date: | 01/2009 |
| Completion Date: | 12/2009 |
| Abstract: |
| Burkholderia sp. MBA4 was enriched from soil using monobromoacetic acid as the sole carbon and energy source. This bacterium contains a haloacid operon, which encodes for a dehalogenase (Deh4a) and an associated permease (Deh4p). The expression of the operon has been found to be regulated. The gene products were detected only in the presence of haloacids and not when the cells were grown in pyruvate-containing or rich medium. The physical and biochemical properties of Deh4a have been well characterized and the investigation of Deh4p has also been initiated. The study on the regulation of the operon, however, is far from sufficient due to intrinsic difficulty to work on the genetics of the natural isolates. We have initiated the study by using a green fluorescent protein (GFPuv) as a reporter and showed that a DNA fragment containing 100 bp of upstream non-coding sequence of the deh4a gene was sufficient for regulated expression. In silico analysis of this fragment has identified the presence of a -35 and a -10 boxes, typical of sigma-70 dependent RNA-polymerase type promoters. The use of overlapping bandshift assay showed that this 100-bp region is necessary and sufficient for the formation of retardation complexes. The biological importance of these -35 and -10 boxes was also confirmed by site-directed mutagenesis and quantitative reverse-transcriptase PCR analysis of a reporter gene. Replacement of the -10 box abolished gene expression completely. Mutation of the -35 box affected the transcriptional efficacy of the promoter. In order to obtain more information on the regulatory protein of this promoter the 100-bp fragment was ligated to form a trimer and used for making of a DNA affinity column. Cell extracts prepared from un-induced cells (grown in LB without NaCl) were fractionated by ammonium sulphate precipitation, Heparin Sepharose column and the DNA-affinity column. Fractions containing the DNA-binding ability were analyzed by SDS-PAGE gel. A 28-kDa protein was purified from the gel and digested with trypsin. The digestion products of this protein were resolved by tandem mass spectrometry and a peptide mass fingerprint was obtained. A peptide with an ion score of 100% confidence interval was also used for comparative analysis with the protein databases. The amino acids sequence of this peptide is FVLTS SVLEL SNGFL R and has been identified as part of a protein from a well-studied polychlorinated biphenyl degrader Burkholderia xenovorans LB400. This orthologous protein in LB400 has been identified as a member of the COG1414, which contains many transcriptional regulators. In this proposal I would like to clone and characterize this transcriptional regulator of the haloacid operon of Burkholderia sp. MBA4. Gene specific oligonucleotide primers will be designed according to the DNA sequences of the regulator genes in closely related species whose genomic sequences are available. These primers will then be used to amplify the corresponding gene in MBA4. The cloned gene will then be expressed in an appropriate Escherichia coli to produce sufficient amount of protein for structural and functional characterization. The expression of this regulator in MBA4 will also be investigated by quantitative reverse-transcriptase PCR to see if it were produced constitutively or in a regulated manner. |
| Project Title: | 34th FEBS Congress Mutagenic analysis of the conserved residues of a haloacid permease |
| Investigator(s): | Tsang JSH |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 07/2009 |
| Completion Date: | 07/2009 |
| Abstract: |
| N/A |
| Project Title: | Purification and characterization of a DNA-binding protein of a dehalogenase producing Burkholderia |
| Investigator(s): | Tsang JSH |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 03/2010 |
| Abstract: |
| Haloacetic acids can be found in the natural environmental because of chemical and biological activities. However, most of the haloacetic acids that we detected nowadays were produced via various human activities such as water disinfection, pesticide production and refrigerant production. Haloacetates are mutagenic and/or cytotoxic and inhibit enzyme activities in the general metabolic pathways. One of the haloacetates, monochloroacetic acid, has been used regularly in the chemical industry for making of human commodities. Monochloroacetic acid affects the citric acid cycle, gluconeogenesis and cause damages to liver and kidney. The indiscriminate use of monochloroacetic acid has causes environmental concerns. Haloacetic acids are also produced as a result of the biodegradation of other halogenated compounds such as methyl chloride and polychlorinated biphenyls. Microorganisms have been recognized as one of the major player in remediating these harmful chemicals. Microorganisms capable of degrading these halogenated compounds usually possess an enzyme, dehalogenase, that cleaves the carbon-halogen bond and mediate the metabolic products utilizable. The genus Burkholderia is a group of microbes with versatile metabolic capability. This group of bacteria has been found in various niches. They have been isolated as animal and plant pathogens. B. cepacia and B. cenocepacia are clinically important species because of their association with cystic fibrosis patients. Some species have been acted as pathogen antagonists and others have been categorized as plant growth-promoting species. Many others have been isolated as important organisms responsible for the bioremediation of environmental pollutants. B. xenovorans LB400 has been well studied as one of the most versatile degraders that metabolize polychlorinated biphenyls. My group has been working on the degradation of halogenated alkanoic acids for many years. A Burkholderia sp. was isolated from soil for its ability to produce a dehalogenase, Deh4a, which degrades 2-haloacids. The enzyme has been purified and the active enzyme has been found to be a dimer of 45 kDa. The gene encoding for this enzyme has been isolated and cloned into Escherichia coli. By means of domain-swap experiments with another dehalogenase, the region responsible for the dimerization of the protein has been identified. Site-directed mutagenesis has also been used to find the residues essential for the structure and activity of the enzyme. A crystal structure of the enzyme has also been resolved. Since haloacetic acids are toxic it would be reasonable to assume the production of the dehalogenase as a detoxifying enzyme and, if necessary, the making of an efflux protein to decrease the availability of the compound inside the cell. During the characterization of the genomic organization of the dehalogenase gene, we have identified the presence of an associated transporter protein gene. This permease, Deh4p, transported the haloacetic acids into the cell and the gene is located downstream of the dehalogenase gene. The gene encoding for Deh4p has been cloned and sequenced. This permease is 552 residues long and has a putative molecular weight of 59,414 and an isoelectric point of 9.14. Comparative analysis of the primary structure of Deh4p with proteins in the protein family (Pfam) database has designated it as a member of the Major Facilitator Superfamily. It looks like the dehalogenase and the permease genes form an operon responsible for the degradation and uptake of the haloacetates into the cell and converted the harmful chemicals to utilizable substrates. In order to confirm that these two genes were organized as an operon the upstream non-coding region of the dehalogenase gene has been explored. This region has the structural features of typical bacterial housing-keeping genes. Traditional -10 and -35 boxes required for the binding of a sigma-70 RNA polymerase were identified. While the expression of the gene, in multiple copies, in E. coli was minimal, it can be induced more than 200-fold by haloacetic acids in Burkholderia sp. MBA4. The gene encoding Deh4a was isolated in a 1.6-kb EcoRI fragment, which include 832 bp of upstream non-coding sequence. When this upstream sequence was truncated, progressively, and used to drive the expression of a reporter gene, it showed that 100-bp of upstream sequence is sufficient for the haloacetate induction mechanism. DNA fragment containing this 100-bp was amplified and labeled with radioisotope for electrophoretic mobility shift assay. Cell extracts were prepared from cells grown on pyruvate or monochloroacetate. The results showed the presence of retardation complex in cell extract prepared from pyruvate-grown cells and not on monochloroacetate-grown cells. This suggested that there is a DNA-binding protein that binds the regulatory region of the dehalogenase gene and prevented it from expressing when there were no haloacetic acids in the medium. This DNA-binding protein is most likely a repressor molecule exhibiting negative effect on the promoter. A proposal has been written and submitted to RGC for purification and characterization of this DNA-binding protein. However, the reviewers would like to see the protein purified and its DNA-binding ability confirmed. In this proposal I would like to ask for funding to purify this DNA-binding protein from MBA4. When the purified protein is available its DNA-binding ability will be confirmed by bandshift assay and the recognition-sequence/region determined by footprinting. The protein will also be subjected to tryptic digest and the digestion products analyzed by tandem mass spectrometry. This would be able to provide some preliminary information of the protein. The immediate goal is obtain sufficient background information on this molecule so that it can be cloned from MBA4 for further characterization. A proposal on the cloning and characterization of this regulatory protein will be submitted to RGC for a GRF grant in 2010. The ultimate aim of the research is to understand the regulatory mechanism for the dehalogenase. |
| Researcher : Wang M |
| Project Title: | Preventive potential and mechanism of fruit phytochemicals on the formation of mutagenic heterocyclic amines |
| Investigator(s): | Wang M |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 09/2007 |
| Completion Date: | 08/2009 |
| Abstract: |
| To identify potent inhibitors for HA formation from pineapple and elderberry extracts using chromatographic and spectroscopic methods and to clarify whether their intervention can really lower overall mutagenicity in meats; to tentatively clarify the roles of several phenolic compounds in the formation of HAs and to elucidate the detailed inhibitory mechanisms involved. |
| Project Title: | Natural tyrosinase inhibitors as skin whitening agents |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Innovation and Technology Fund Internship Programme |
| Start Date: | 10/2007 |
| Abstract: |
| The basic aim of this study is to develop patentable tyrosinase inhibitors (purified compounds, standardized concentrated plant extracts) from edible and Chinese medicinal plants, and evaluate their appliction as skin whitening/lightening agents in cosmetic products. |
| Project Title: | Natural Phenolics for Ameliorating Carbonyl Stress |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 04/2008 |
| Completion Date: | 09/2009 |
| Abstract: |
| Non-enzymatic modifications of proteins have been implicated in the pathogenesis of diabetes, atherosclerosis, and neurodegenerative diseases as well as in normal aging. The modifications can arise from direct exposure to reactive oxygen related species, chlorine, nitrogen species and from reactive with low molecular weight reactive carbonyl compounds, which originate from a multitude of mechanistically related pathways, like glycation, sugar autoxidation, lipid peroxidation and uv-photodamage. The accumulation of various reactive carbonyl species derived from either carbohydrate or lipids, as well as their subsequently induced protein modifications is proposed to constitute a state of “carbonyl stress”. It’s well known that a group of dicarbonyl compounds, including 3-deoxyglucosone (3-DG), glyoxal (GO) and methylgoxal (MGO) will be formed in Maillard reaction (glycation). The increased electrophilicity of these 1,2-dicarbonyl compounds results in their relatively fast reactions with amino, sulfhydryl and guanidine functional groups of intracellular and extracellular proteins. Lipoxidation mainly generates a burst of unsaturated aldehydes, such as 4-hydroxy-trans-2-nonenal (HNE), acrolein (ACR), and malondialdehyde (MDA). Meantime lipoxidation is also known to produce GO. In contract to glycation-related carbonyl compounds, less is known about protein modification by lipoxidation-derived RCS. However a large body of evidence indicates that many of the effects of vascular dysfunction on cardiovascular diseases are mediated by lipid-derived RCS and the emerging role of lipid-derived RCS in hyperglycemia and in development of complication of diabetes is now well documented. Recently, the involvement of lipid-derived RCS as products and propagators of oxidative damage in neurodegenerative diseases, mainly AD, is also becoming elucidated. Under carbonyl stress, not only advanced glycation end-products (AGEs) derived from carbohydrates but also advanced lipoxidation end-products (ALEs) derived from lipid accumulate in parallel. AGEs and ALEs might be merely end-results of protein structural modifications, alternatively they might play an active role in the development of various age-related diseases. In addition, compared to free radicals, RCS are stable and can diffuse within and even escape from cell and attack target far from the site of the original formation. Therefore they are not only end-products of glycation/lipoxidation, they also act as “second cytotoxic messengers”, induce different aspects of cellular stress. Thus there is a need to develop strategy to deal with carbonyl stress, to ameliorate various age-related diseases. A very limit number of inhibitors of cellular reactive carbonyl species have been identified to date and their therapeutic potential are recognized only recently. Some inhibitors interfere with the reaction by trapping carbonyl compounds, while others act merely as antioxidants and transition metal chelators. As free radicals and oxidation are known to be in the process of glycation and lipoxidation, it’s not a surprise that antioxidants may be effective in in vitro assays. However it’s questionable of the effects of antioxidant against carbonyl stress, AGE and ALE formation in the real biological system. As an example, the capability of pyridoxamine (PM) to delay development of diabetic complications in animal models has been well documented. The biological effects of PM, notably its inhibition of development of renal and vascular disorders in both diabetic and Zucker rats, may be chiefly attributable to its function as an inhibitor of AGE/ALE formation through a carbonyl-trapping mechanism. Although PM is known to be an antioxidant, it has been shown to be incapable of preventing oxidative loss of linoleate or arachidonate in phosphate buffer, even at 1mM concentration. On the other hand, it can significantly inhibit the formation of CML, CEL and MDA-Lys, and of HNE-Lys (2-hydroxylhexanal-lysine complex) in the presence of ribonuclease, suggesting that it does not function primarily as an antioxidant. It is an attractive hypothesis that antioxidation may not be the dominant action mechanism of other compounds found to have inhibitory activity against AGE formation. In addition, numerous clinical trials have failed to provide conclusive evidence for the efficacy of antioxidant therapy in several chronic diseases, questioning the effects of antioxidant against carbonyl stress. Thus the chemical agents which can directly trap reactive carbonyl species will be more promising and have real clinical application. Natural products have been shown to be safer for human consumption than synthetic compounds. In this regard, some plant extracts have been evaluated for their inhibitory effects on reactive carbonyl induced protein structural modification. However, only a very limited number of natural products have been found to have reactive carbonyl-trapping capacity. Lo et al. in 2006 reported the MGO trapping reaction of green tea catechins and black tea theaflavins under simulated physiological conditions. The reaction products of EGCC and methylglyoxal were separated by chiral column and the structures were confirmed by 2D-NMR analysis. This study provided the first solid evidence that certain groups of phenolic antioxidants can directly trap reactive carbonyl species, and compelled a reconsideration of the anti-glycation mechanism of phenolic antioxidants. The findings suggested that certain phenolics possessing dual mechanisms of action, namely antioxidation and RCS scavenging, maybe potentially more effective in preventing reactive dicarbonyl compounds induced protein glycation. Our recent research also found procyanidin B2 can effectively trap MGO, with better trapping capacity than aminoguanidine (AG), the first AGE inhibitor explored in clinical trials. Careful examining the structures of catechins, procyanidin B2, we found they contained unique nucleophilic moiety which might contribute to their MGO trapping capacity. However little is known whether other types of phenolic compounds can trap MGO (an intermediate carbonyl compound produced during glycation) or not, and whether phenolic compounds can direct trap lipoxidation-related unsaturated carbonyl compounds or not. All these demand further scientific clarification. The basic aims of this study are to elucidate the RCS trapping capacities of various phenolic compounds, evaluation their trapping mechanism through detection of conjugated compounds and structural elucidation, and evaluation of their effects on the formation of AGEs and ALEs. In brief, the study seeks to make a significant contribution to the amelioration and prevention of several chronic diseases by discovery of specific phenolic RCS-trapping agents. |
| Project Title: | Citrus Flavonoids as Food Additives for Reducing the Formation of Hazardous Substances in Processed Food Products |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Applied Research |
| Start Date: | 12/2008 |
| Completion Date: | 12/2009 |
| Abstract: |
| A large portion of our disposable income is spent on food, only second to housing. Foods provide humans essential nutrients for growth and health maintenance. On the other hand, it is estimated that up to 50% of cancers are diet related. The potential harmful effects of foodborne toxicants have been well recognized. In addition to the well-known hazardous substances such as pesticides, heavy metals, antibiotics, environmental contaminants in foods, a group of compounds including heterocyclic amines (HAs), polycyclic aromatic hydrocarbons (PAHs), reactive carbonyl species (RCS), advanced glycaion end products (AGEs) and akrylamide might also contribute to the mutagenicity, carcinogenicity and other toxic effects of certain food products. These compounds can be regarded as by-products of food heat processing, such as roasting, baking, broiling and frying, which are essential for food safety and improvement of organoleptic properties of foods. Further studies showed that fundamental food components including carbohydrates, amino acids, proteins and lipids undergo degradation, oxidation, caramelization and/or interact through Maillard reaction and various adduction reactions, giving rise to these toxicants. Mutagenicity/carcinogenicity of many of these compounds has been established. The good side is that there have been continuous research approaches aiming to reduce the formation of these compounds in foods. In the past, a combination of temperature, pH, cooking time, water activity and precursor compositions/concentrations were central determinants for the final toxic products formed. In other words, these have been targets for developing inhibitory strategies. However, the viewpoint has changed ever since the growing popularity of incorporating various food additives, plant extracts or tissues into different cuisines and the realization of the importance of natural antioxidants in food systems. A variety of synthetic and natural agents have been examined using both simple chemical model and real food systems. The major pitfall is that most of these inhibitors were tested only for their activity against the formation of a very limited number of genotoxic substances. Considering the fact that the above mentioned classes of toxicants are formed from similar primary food components and that the reaction pathways, though differ, may be concurrent under many food processing conditions, it is believed that intervention with one of these pathways would have significant impact on the chemistry of others. This led us to initiate the research effort to identify natural additives which are active against the formation of a wide spectrum of toxic compounds formed during of thermal preparation of foods, thus minimizing the overall toxicity load of the final food products. Our recent basic research work supported by RGC has demonstrated that certain phytochemicals can significantly inhibit the formation of mutagenic HAs in foods. One of them, naringenin, which widely occurs in citrus fruits and other plants such as tomato and apple, was found to be especially potent. Our mechanistic study has brought a new insight into the mechanism of inhibition by natural phenolic compounds, whose inhibitory mechanisms were frequently attributed to antioxidation by others. In other words, we proved that alternative key inhibitory mechanism(s) exist and the findings have been published in several top journals in the field of food science and technology. We currently has got a breakthrough discovery that naringenin probably inhibits formation of HAs, in particular, PhIP (the most abundant HA in foods) by directly trapping RCS which are critical HA intermediates. However, much further research has to be continued to realize our ultimate goal of the project – development of a commercial food additive with inhibitory activity against a broad spectrum of toxic compounds in processed foods. This will include evaluating the effect of potential flavonoids, especially naringenin, on the formation of other types of hazardous substances in foods, identification of synergistic or counteracting phytochemicals in the citrus extracts of interest, processing of by-products from citrus juice production into extracts which, ideally are to be selectively concentrated in favor of the principal inhibitors as well as synergistic components, and finally assessment of the effect of such additive on the sensory properties of the food products examined. It is hoped that commercialization of such natural extract would contribute significantly to reduction in human exposure to food-borne toxicants. The specific aims of this project are: Objective 1: Development of sensitive analytical methods for analysis of PAHs, RCS, akrylamide, and AGEs in model systems and real food products. Objective 2: Preparation of different citrus extracts using different technologies and evaluating the effects of these extracts on formation of toxic substances in processed foods. Objective 3: Development of citrus extract(s) of interest into commercial food additive which is selectively concentrated in favor of potent inhibitors identified. |
| Project Title: | The 237th ACS National Meeting & Exposition Tyrosinase inhibitors from Artocarpus heterophyllus as antibrowning agents for apple slices |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 03/2009 |
| Abstract: |
| N/A |
| Project Title: | Integrated approaches to evaluate the bioactivities of process-induced novel flavonoid derivatives in thermally processed food systems |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 06/2009 |
| Completion Date: | 11/2010 |
| Abstract: |
| Almost all food items consumed are processed to some extent. Thermal treatment is among the most popular ways of processing. During heating, a complex array of chemical reactions take place which play a pivotal role in determining the quality attributes (sensory, nutritional and safety) of the process foods. While some compounds are destroyed during food processing, many more new compounds might be introduced into the food system. Some of these compounds contribute significantly to the organoleptic properties of foods, such as color and flavor. On the other hand, compounds with various biological activities could also be resulted from the heat-induced reactions. Some of them have been shown to be potential antioxidative and chemopreventive agents. In contrast, some are harmful and might pose significant health risks for human beings in the long term. Representative members of such food-borne toxicants include heterocyclic amines (HAs), polycyclic aromatic hydrocarbons (PAHs), reactive carbonyl species (RCS), advanced glycaion end products (AGEs) and acrylamide. The formation of these compounds involves complex networks of reactions, though many start with fundamental food components such as glucose, amino acids/proteins, and lipids. Other ingredients such as flavonoids may interact with these reactions. Flavonoids, in the broad sense, are virtually universal plant pigments. They are responsible or partially responsible for the color of flowers and sometimes leaves. When they are not directly visible, they contribute to coloration by acting as copigments. Flavonoids have a common biosynthetic origin and possess the same basic structural element, namely the 2-phenylchromane skeleton. They fall into about a dozen classes depending on the degree of oxidation of the central pyran rings; best known flavonoid skeletons are flavone, flavonol, flavanone, flavan, anthocyanin and chalcone. Most of these skeletons (with the exception of anthocyanin) are quite stable. Flavonoids have been well known for their antioxidant activity. Recently, they have been recognized as a class of natural products which could be utilized to fortify various food products with powerful antioxidants either for health benefits or pure marketing strategy. In addition, flavonoids or plant extracts with high concentration of flavonoids are quite popular as novel food additives for food products to reduce the formation of mutagenic and carcinogenic heterocyclic amines (HAs), polycyclic aromatic hydrocarbons (PAHs) and acrylamide by interaction with one key chemical reaction in food science, Maillard reaction. Our former research work has demonstrated that two flavonoids, naringenin and EGCG can significantly inhibit the formation of mutagenic HAs in foods. Our mechanistic study has also brought a new insight into the mechanism of inhibition of HA formation by flavonoids, whose inhibitory mechanisms were frequently attributed to antioxidation. We currently have got a breakthrough discovery that naringenin and EGCG mainly inhibits the formation of HAs, in particular, PhIP (the most abundant HA in foods) by directly trapping Strecker aldehyde, phenylacetaldehyde which is a key intermediate PhIP formation. Novel adducts are formed between the reaction of flavonoids and phenylacetaldehyde. In case of naringenin, inhibition of PhIP formation gave rise to 8-C-(E-phenylethenyl)naringenin and 6-C-(E-phenylethenyl)naringenin in high concentrations. Independent of this, we also discover that phloridzin, a chalcone-type flavonoid and epicatecin, a flavan can effectively trap methylglyoxal, another key Maillard reactive intermediate even at room temperature to form some novel adducts. All these findings suggest that certain flavonoids could directly participate in the Maillard reaction (the reaction between sugar and amino acid/proteins, the most important chemical reaction in food), and consequently lead to the formation of flavonoid derivatives that are structurally distinct from the parent flavonoids. It is therefore anticipated that these derivatives might have distinctive bioactivities, too. In addition, drastic heat treatment could also cause degradation of flavonoids, thus introducing further potentially bioactive compounds into the food systems concerned. Many of them will likely remain in the food products and be taken into the human body upon consumption of the food products. Ideally, these newly formed compounds could confer beneficial effects, such as enhanced antioxidant, and antimutagenic/anticarcinogenic activities to human beings. Yet, it is probable that some of these derivatives are toxic. The impact of these thermal treatment-induced derivatives of natural products should by no means be neglected. Surprisingly, this area of enormous significance to food quality remains largely untouched. In the current study, we plan to address the above issues from a multitude of approaches: chemical, molecular pharmacological, and metabolomics approaches. With advanced analytical tools in place, we aim to characterize key thermal treatment-induced derivatives of flavonoids as well as to evaluate their biological activities. Specific objectives of this research work include: 1. Metabolomics approach to evaluate the chemical profile of the selected flavonoids in food system. Isolation and structural characterization of thermal process-induced degradation products of these flavonoids and new derivatives formed from the reaction between these flavonoids with other food ingredients, particularly formed in a few of important Maillard reaction chemical systems (glycine with glucose, phenylalanine with glucose, asparagine with glucose, and tryptophan with glucose). 2. Evaluation of the mutagenicity and toxicity of the newly formed derivatives (adducts) or degradation products of flavonoids in different Maillard reaction systems. 3. Evaluation of antioxidant and anti-carcinogenic activities of the newly formed adducts. Their selective cytotoxicity in normal versus cancer cells will be examined, and potential anti-cancer mechanisms will be tackled with molecular approaches. |
| Project Title: | American Chemical Society Fall 2009 National Meeting & Exposition Dietary phenolics: New roles in disease prevention and food application |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | URC/CRCG - Conference Grants for Teaching Staff |
| Start Date: | 08/2009 |
| Completion Date: | 08/2009 |
| Abstract: |
| N/A |
| Project Title: | Development of Novel Skin Whitening Cosmetic Products with Natural Tyrosinase Inhibitors as Key Ingredients |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Innovation and Technology Support Programme (Tier 2) |
| Start Date: | 04/2010 |
| Abstract: |
| The basic aim of this study is to develop patentable tyrosinase inhibitors (purified compounds, standardized concentrated plant extracts) from Chinese medicinal plants, evaluate their application as skin whitening/lightening agents in cosmetic products, and develop cosmetic formulas with these novel ingredients as key ingredients. The specific objectives are: Objective 1. To develop a highly purified and concentrated Artocarpus heterophyllus extract, with the overall content of steppogenin, artocarpesin, norartocarpetin, artocarpanone, and isoartocarpesin to reach 20%. Objective 2. To isolate, purify and elucidate the structures of tyrosinase inhibitors from Morus australis root extract and Cudrania tricuspidata twig extract and evaluate their effects on melanin synthesis and cell viability in melan-a cell culture. Objective 3. To formulate Artocarpus heterophyllus extract into real cosmetic products (cream, lotion, serum and paper mask) and test their safety, and efficacy. |
| Project Title: | Establishing a Quality Control Platform for Popular Nutraceutical Products in Hong Kong Market |
| Investigator(s): | Wang M |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | Seed Funding Programme for Applied Research |
| Start Date: | 06/2010 |
| Abstract: |
| The term "nutraceuticals" is a combination of the words nutrition and pharmaceuticals, and refers to substances which possesses physiological benefits or provides protection against chronic diseases. Nutraceuticals may include fortified foods, functional foods, specific diets, genetically engineered foods, herbal products, dietary supplements, and processed foods such as cereals, soups, and beverages. Specific examples of nutraceuticals are resveratrol from red grape products as an antioxidant, soluble dietary fiber products, such as psyllium seed husk for reducing hypercholesterolemia, broccoli (sulforaphane) as a cancer preventive agent, soy or clover (isoflavonoids) to improve arterial health, and vitamins and minerals for general health management. In recent years, consumers have become more health conscious. The concept of taking nutraceuticals to promote health and prevent diseases such as heart disease, cancer, osteoporosis, arthritis, and type 2 diabetes mellitus is widely recognized. As a consequence, there has been a rapid increase in the demand for nutraceuticals which has been the major driving force for the development of nutraceutical products to suit the need of different groups of customers. The nutraceutical industry is booming and the sales of for the global nutraceutical industry is over 180 billion USD annually. There are different types and forms of nutraceuticals on the market. As for different commercial purposes, there are different quality control and quality assurance standards. But usually they follow some general rules, the raw materials must be authenticated, safe to use, with a limit of foreign materials, heavy metals, aflatoxins and pesticides. The pH value, ash contents, moisture contents and particle size should be in a reasonable range. Also the microbiological tests must be passed. The general quality control standards are usually able to be met. But specific standards for nutraceutical products are usually lacking, and this is one of the major challenges that face the nutraceutical industry. Instrumental methods, especially HPLC (high pressure liquid chromatography) are well known to be good methods for quality control and fingerprinting nutraceutical as well as pharmaceutical products. HPLC coupled with photodiode array detector, MS/MS and ELSD (Evaporative light scattering detector) are suitable for the analysis of various nonvolatile components in nutraceutical products and GC (Gas Chromatography) and GC/MS are good for analysis of volatile compounds, fatty acids and sterols. By careful development and validation, these instrumental methods can be used to systematically control the quality of various nutraceutical products. A recent market research pointed out that based on broad applications and increasing clinical evidence of health benefits and safety of nutraceuticals, the following types of products will likely have the best growth opportunities: soy protein nutrients; lutein, lycopene, omega-3 fatty acids, probiotics and sterol esters which can easily used as functional food and beverage additives; essential minerals calcium and magnesium; herbal extracts, garlic (for improving cardiovascular functions), green tea (for cancer prevention and weight loss), bilberry (for eye health), saw palmetto (for benign prostatic hyperplasia) and black cohosh (for postmentopausal symptoms); and the non-herbal extracts chondroitin, glucosamine and coenzyme Q10. In addition, global demand for nutraceutical vitamin ingredients will continue to increase. These products have already been very popular in Hong Kong and Mainland China. Together with classic Chinese healthy foods such as Reishi mushroom, bird nest, these products are the key healthy products in Hong Kong. However, in Hong Kong, we are lacking of quality control procedures and methods for these nutraceutical products, there is no lab which can perform and provide quality control analysis for them although they are very popular products in the market. This is different from the status of traditional Chinese herbal medicines, for which the government has invested a lot of resources to develop quality control procedures and this scheme has been one of major strategies for modernization of traditional Chinese herbal medicines. In this project, we want to establish a quality control platform for nutraceutical products. A survey of popular nutraceutical products in Hong Kong will be conducted, and methods will be developed and validated for the quality control of these products. These methods can be adopted by governmental agencies for regulation of nutraceutical products in HK, by local nutraceutical companies for production of high-quality nutraceutical products, and by the commercial testing labs in Hong Kong to provide service to local and international companies in the nutraceutical business. |
| Researcher : Yip WK |
| Project Title: | Functional characterization and subcellular localization of three ethylene receptors in rice |
| Investigator(s): | Yip WK, Yau CP |
| Department: | Botany |
| Source(s) of Funding: | Seed Funding Programme for Basic Research |
| Start Date: | 01/2005 |
| Abstract: |
| Ethylene is an important plant hormone which regulates a range of developmental and physiological process in plants. In the past decades, exceptional progress has been made on understanding the molecular mechanism controlling the ethylene signalling pathway which was shown to require membrane-associated ethylene receptors to function. In Arabidopsis, there are altogether five ethylene receptors which negatively regulate ethylene responses. However, little is known about the involvement of receptor genes in the ethylene perception of monocotyledonous plants such as rice. Recently, we have successfully isolated five putative ethylene receptor genes from rice and showed that their expression levels were regulated developmentally, and by various external stimuli including ethylene. Here we propose to functionally characterize three rice ethylene receptor genes, OS-ERS1, OS-ERS2 and OS-ETR2 in planta. Moreover recent studies on the subcellular localization of two ethylene receptors, At-ETR1 and NtHK1 (an ethylene receptor ortholog in tobacco) revealed that they were localized to endoplasmic reticulum and plasma membrane respectively. To understand more in detail how these three rice ethylene receptors function in cellular level, we propose to investigate their corresponding subcellular localization by fusing with fluorescent protein. |
| Project Title: | Characterization of a cyanide detoxification gene encoding L-3-cyanoalanine synthase (CAS), and determine its roles among the cysteine synthase (CS)/CAS family in the rice genome |
| Investigator(s): | Yip WK |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2007 |
| Completion Date: | 12/2009 |
| Abstract: |
| (1) Study of OS-CAS transcription by promoter analyses; (2) heterologous expression of OS-CAS in bacteria dnd yeast to test its catalytic activity; and unravel the structure and function relationships between CS and CAS by site directed mutagenesis studies; (3) subcellular localization of OS-CAS by tagging it with the yellow fluorescent protein (YFP); (4) the roles of OS-CAS and CS on cyanide detoxification and cysteine synthesis in rice; to investigate whether quincorac or 2,4 D resistant in rice is conferred by OS-CAS. |
| Project Title: | The study of plant hormone interactions on growth, development and physiological responses using ACC synthase gene suppression mutants in tomatoes. |
| Investigator(s): | Yip WK |
| Department: | Botany |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2008 |
| Completion Date: | 12/2009 |
| Abstract: |
| (1) Elucidation the interactions among ethylene, abscisic acid, and IAA on stomatal aperture and transpiration. (2) Elucidation the interactions among ethylene, abscisic acid, and IAA on seedling growth. (3) Study on the possible hormonal interactions between ethylene and ABA in seed development in tomato fruits. (4) Gene discovery on important plant processes by employing the RNA micro-array analyses. |
| Project Title: | Molecular studies on two components OASS and SAT of rice cysteine synthase complex and cysteine regeneration during ethylene biosynthesis |
| Investigator(s): | Yip WK |
| Department: | School of Biological Sciences |
| Source(s) of Funding: | General Research Fund (GRF) |
| Start Date: | 01/2009 |
| Abstract: |
| (1) To have a basic understanding of the gene expression profiles of the OASS/CAS gene family members during seedling growth in light and in dark, and also under the ethylene/auxins treatments; (2) To find out the subcellular localization of individual OASS/CAS members; (3) To verify the functions of OASS/CAS, and SATs encoded by these two gene families by complementation expression in bacteria or yeast mutants; (4) To elucidate the CS complex formation of gene products encoded by these two gene families and find out whether hetero-subunits formation would be possible; (5) To determine whether cysteine synthesis would be possible in rice mitochondria or cysteine has to be imported from cytosol during active ethylene biosynthesis. |
| Researcher : Yu KY |
| List of Research Outputs |
| Lo C.S.C., Yu K.Y., Shih C.H., Du Y. and Chu H., Molecular dissection of pathogen-inducible flavonoid pathway in sorghum, XIV International Congress on Molecular Plant-Microbe Interactions. 2009. |