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
Its 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 [Link]
From Yeast
Rate limiting enzymes for DXP pathway
Exists in [Link]
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!
