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Research Exercise Summary

Conducted by: Prabhat Khanal Research exercise mentor: Dr. Hagai Abeliovich
INTRODUCTION Metabolic engineering is the alteration of cellular activities by the manipulation of enzymatic, transport and regulatory functions of the cell by using recombinant DNA technology. Biosynthesis of natural products such as many secondary metabolites by using microorganisms is a new area of metabolic engineering. Secondary metabolites are compounds produced by plants that are not involved in basic metabolic processes or are not directly essential for the photosynthetic or respiratory metabolism in plants. Now, many plant secondary metabolites have been identified to have health promoting and beneficial effects on human health and new roles of such compounds on human health are being found. Difficulty to obtain sufficient amounts of desired plant, slow growth of plants, varying composition and concentration depending upon the geographical position and climatic conditions and low yield of isolated compounds are some of the limitations of commercial extraction of these compounds by using plant as a single resource. On the other hand, some of the bottlenecks of chemical synthesis may include higher energy requirements, pollution, low reaction load due to unwanted chemical reactions, cost and availability of starting materials and cost of separating and purifying the end products. Therefore, metabolic engineering of microorganisms like yeast through the heterologous and functional expression of foreign genes encoding many plant secondary metabolites of great importance on human health could be an important alternative for the successful production of such compounds. OBJECTIVES
• To give an overview of various works that have been done in the field

of metabolic engineering of plant secondary metabolites by using yeast and to search the possibility of using the yeast as an efficient cell factory for the production of various human health promoting plant secondary metabolites in the coming future.

antiviral and antibiotic properties. antitumour. plant secondary metabolites can be divided into three major groups: (a) flavonoids and other polyphenolic compounds (b) isoprenoids (terpenoids) compounds (c) nitrogen containing alkaloids and sulphur containing compounds. cinnamate-4-hydroxylase (C4H). Flavanone has been successfully produced in yeast by expressing phenyl ammonia lyase (PAL). Ro et al. Production of Flavonoids in Yeast Flavonoids are produced in yeast by expressing phenylpropanoid pathway. is synthesized by the cyclization of farnesyl pyrophosphate (FPP). cerevisiae to produce high amount of artemisinic acid using an engineered mevalonate pathway. Many flavonoid compounds are successfully produced in yeast by cloning genes from different plant species and microorganisms. some of them are interesting and extremely important compounds in human health because of their potent anticancer. a sesquiterpene to precursor to the antimalarial drug artemisinin. (a) Production of monoterpenoids in yeast: Oswald et al.. only MVA pathway is involved in the biosynthesis of ergosterol as the major end product. rate limiting steps and enzymes involved. Flavones have also been produced in flavanone producing recombinant yeast by expressing flavone synthase I (FSI) and flavone synthase II (FSII) genes. Production of Terpenoids in Yeast The biosynthesis of terpenes in higher plant cells shows two entirely separate enzymatic pathways: mevalonic acid pathway (MVA) and methylerythritol 4-phosphate (MEP) pathway. it is necessary to know the biosynthetic pathways of those compounds. (2007) engineered yeasts to produce monoterpenols by expressing linalool synthase and geraniol synthase genes and yeast strains successfully produced those monoterpenoid alcohols by using internal geranyl pyrophosphate (GPP).• To understand the mechanism behind the metabolic engineering of yeast for the production of plant secondary metabolites that have beneficial and health promoting effects on humans. For the metabolic engineering of plant secondary metabolites. cytotoxic. (2007) used S. Literature Review Based on biosynthetic origins. (b) Production of sesquiterpenes in yeast: Sesquiterpenes are the most diverse class of isoprenoids. In yeast. amorphadiene . Amorphadiene. 2005). 4-coumarate-CoA (4CL) and chalcone synthase (CHS) genes (Yan et al.

(2006) cloned a gene encoding geranylgeranyl pyrophosphate synthase (GGPPS) from bell pepper (Capsicum annum) in S. a non-carotenogenic yeast. In future. reducing flux through competitive pathways. utilities of different yeast species should be investigated in future for the efficient microbial production of such compounds.synthase (ADS) and a novel cytochrome P450 monooxygenase from Artemisia annua. (2001) expressed genes coding for strictosidine synthase (STS) and strictosidine glucosidase (SGD) enzymes from medicinal plant Catharanthus roseus in Saccharomyces cerevisiae and successfully produced cathenamine from tryptamine and secologanin by functionally expressing those two enzymes in yeast. commercially valuable and nutritionally important plant secondary metabolites compounds. REFERENCES . CONCLUSION This study clearly shows that yeast is an important and attractive host for the heterologous and function expression of foreign genes encoding many plant secondary metabolites of great importance on human health. coli also have been used for the production of some of the plant secondary metabolites but similar intracellular compartments. Along with the increased knowledge on biosynthetic pathways of many plant secondary metabolites. (c) Production of carotenoids in yeast: Schizosaccharomyces pombe. Gunel et al. reducing catabolism and overexpression of regulatory genes are some of the strategies that can be used for increased secondary metabolites production through metabolic engineering. capability for the posttranslational modifications of an eukaryotic protein and ability to express cytochrome P450 enzymes have provided yeast as a suitable host for the expression of complex eukaryotic pathways. metabolic engineering of microorganisms like yeast can be an alternative production system to overcome the limited availability of biologically active. Production of Plant-Origin Alkaloids in Yeast Geerlings et al. S. pombe and successfully redirected carbon flow from the terpenoid pathway leading to ergosterol formation towards the production of carotenoid through the heterologous expression of carotenoid biosynthetic gene in a non-carotenogenic yeast. is not able to produce any carotenoids but it synthesizes ergosterol from FPP through the sterol biosynthetic pathway. Overcoming rate limiting steps. pombe. DISCUSSION Although prokaryotic organisms like E.

Appl Environ Microbiol 2005.56:420-4. J Appl Microbiol Biotechnol 2001. Contin A. Dirninger N. Erturk S. Fischer M. . Paradise EM.20:76-82. Oswald M.71:5610-3. Memelink J. Ouellet M. Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast. Koffas MAG.Geerlings A. Heijden RV. Gunel T. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. et al. Verpoorte R. FEMS Yeast Res 2007. Metabolic engineering for production of geranylgeranyl pyrophosphate synthase in non-carotenogenic yeast Schizosaccharomyces pombe. Arda N. Production of the antimalarial drug precursor artemisinic acid in engineered yeast.7:413-21. Kohli A. Nature 2007. Yan A. Karst F. Kuntz M. Biotechnol Biotechnol Eq 2006. Redondo FJ. Temizkan G.440:940 – 4. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. Ro DK.