max metabolicengineering

8/13/2019 Max MetabolicEngineering

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Metabolic Engineering

Max Itkin


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Outline of the Talk

Introduction to Metabolic Engineering

Metabolic Engineering in Plants

Metabolic Engineering in Bacteria


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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.


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Several ways to metabolically

engineer an organism

Block a metabolic flux (re-channel)

Channel a metabolic flux into new cellcompartments

Induce a metabolic flux (can lead to

unexpected results) Introduce a new metabolic pathway into

organism (the most successful way)


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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 (5.00025.000 per plant)


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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 ofexperiment) 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


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Monsanto Co. has agreed to

provide royalty-free licenses to

speed up work on a genetically

modified rice that could solvevitamin A deficiency around the

world

NEWS OF THE WEEK

Science 11August 2000:Vol. 289. no. 5481, pp. 843 - 845


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Introducing a new pathway into anorganism


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Engineering the Provitamin A)b-Carotene)

Biosynthetic Pathway into (Carotenoid-Free)

Rice EndospermXudong 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


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Engineering theb-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


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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 lcygene.

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


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Results


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Changing Subcellular localizationof an Enzyme


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Science, 2005* Recruitment of biological pest control agents bymetabolic engineering


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The predators choose to go to DMNT /

Nerolidol emmiting plants


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High yields of metabolites in comparison to

plants

Possible to use simple and inexpensive carbonsources (e.g. glycerol)

Fast growing organism (bioreactors)

Codon usage: must use Host preferred codon

usage

Metabolic Engineering in Bacteria


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Introducing a new pathway into an

organism and Inducing a metabolic

flux


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Engineering a mevalonate pathway in

Escherichia colifor 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 itaccumulates iron. It is vulnerable to the oxygen-based free

radicals released by a powerful antimalarial drug known as

artemisinin


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Engineering the biosynthesis of

Artemisinin inE.coli

Rate limiting enzymes forDXP pathway

FromYeast

Exists in

E.coli


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The Main Points of the Article

IPP and DMAPPuniversal precursors to all

isoprenoids, Two pathways exists

FPP biosynthesis and notthe ADS expression was

the rate limiting step

Codon usage: must use Host preferred codon usage

ADS expression raised 10300 times !!!


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Thank you!

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