Metabolic Engineering

Metabolic Engineering Max Itkin Outline of the Talk  Introduction to Metabolic Engineering  Metabolic Engineering in Plants  Metabolic Engineering in Bacteria Introduction to Metabolic Engineering  The use of term “engineering” implies that there is some precise understanding of the system, that is being modified   Rate-limiting steps must be known A typical metabolic-engineering approach focuses on a particular metabolic intermediate or product such as starch, vitamin E, carotenoids, amino acids etc. Several ways to metabolically engineer an organism     Block a metabolic flux (re-channel) Channel a metabolic flux into new cell compartments Induce a metabolic flux (can lead to unexpected results) Introduce a new metabolic pathway into organism (the most successful way) Metabolic Engineering in Plants  Plants are the most prolific factories for small molecules  More then 100.000 metabolites have been identified at 2004 (this may be only 10% of total(  Plants are the richest with secondary metabolites among different organisms (5.000 – 25.000 per plant)  The most successful metabolic-engineering approaches are those that have introduced new pathways into plants e.g. production of provitamin A in rice  It’s a matter of great importance to perform a metabolite profiling of transgenic plants (even in case of failure of experiment) in order to diagnose the problem  Unexpected results can happen e.g. dwarf tomato plants instead carotenoid rich plants  Commercial projects must characterize their plants in order to release plant into a market Monsanto Co. has agreed to provide royalty-free licenses to speed up work on a genetically modified rice that could solve vitamin A deficiency around the world Science 11 August 2000: Vol. 289. no. 5481, pp. 843 - 845 NEWS OF THE WEEK Introducing a new pathway into an organism Engineering the Provitamin A)b-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm Xudong Ye, Salim Al-Babili, Andreas Kloti, Jing Zhang, Paola Lucca, Peter Beyer, Ingo Potrykus * Vitamin A deficiency causes symptoms ranging from night blindness to those of total blindness * In Southeast Asia, it is estimated that a quarter of a million children go blind each year because of this nutritional deficiency * It is estimated that 125 million children worldwide are deficient in vitamin A * Oral delivery of vitamin A is problematic mainly due to the lack of infrastructure Engineering the b-Carotene Biosynthetic Pathway into Carotenoid-Free Rice Endosperm  No rice cultivars produce provitamin A in the endosperm therefore recombinant technologies rather than conventional breeding are required  Immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate, which can be used to produce the uncolored carotene phytoene by expressing the enzyme phytoene synthase (PSY) in rice endosperm  The synthesis of b-carotene requires the complementation with three additional plant enzymes : phytoene desaturase (PDS) and z-carotene desaturase (ZDS), each catalyzing the introduction of two double bonds, and lycopene b-cyclase, encoded by the lcy gene.  To reduce the transformation effort, a bacterial carotene desaturase ,(crt1) capable of introducing all four double bonds required, was used   Transit peptide was attached to crt1 A transit peptide exists in plant PSY Results Changing Subcellular localization of an Enzyme * Recruitment of biological pest control agents by metabolic engineering Science, 2005 The predators choose to go to DMNT / Nerolidol emmiting plants Metabolic Engineering in Bacteria     High yields of metabolites in comparison to plants Possible to use simple and inexpensive carbon sources (e.g. glycerol) Fast growing organism (bioreactors) Codon usage: must use Host preferred codon usage Introducing a new pathway into an organism and Inducing a metabolic flux Engineering a mevalonate pathway in Escherichia coli for production of terpenoids Vincent J J Martin, Douglas J Pitera, Sydnor T Withers, Jack D Newman1 & Jay D Keasling Nature Biotechnology 21, 796 - 802 (2003) 1 June 2003 • The malaria parasite develops inside red blood cells, where it accumulates iron. It is vulnerable to the oxygen-based free radicals released by a powerful antimalarial drug known as artemisinin Engineering the biosynthesis of Artemisinin in E.coli From Yeast Rate limiting enzymes for DXP pathway Exists in E.coli The Main Points of the Article  IPP and DMAPP – universal precursors to all isoprenoids, Two pathways exists  FPP biosynthesis and not the ADS expression was the rate limiting step  Codon usage: must use Host preferred codon usage – ADS expression raised 10 – 300 times !!! Thank you!