IdeaLab this Saturday!

[Nina DiPrimio]

Hey folks -

As requested we are going to talk about metabolic engineering a little bit - specifically the iGEM MIT 2006 Minty E.coli. http://openwetware.org/wiki/IGEM:MIT/2006/Blurb.

There is also a radiolab on the topic:

http://www.npr.org/templates/story/story.php?storyId=90014997.

I am going to give a little background and then open for discussion so we can think about tackling our own metabolic engineering project - whether it is replicating this or doing something similar. 

It would be AWESOME if you have time to do a little literature digging and come up with something you would like to engineer into e.coli. For ease of analysis I would pick something that gives an obvious phenotypic change - new scent (rose) or color? This would require answering the questions - does E.coli contain a precursor to your compound of interest? And if so, is there an enzyme or enzymes in another organism that will convert the precursor to your target compound?

Bring some ideas and we can start putting together a list of parts that fit into the goldenbraid scheme for metabolic engineering! Also, if someone has played around with the metabolic engineering software tools and wants to give a mini demo, please do so!

I will be skyping in.

Look forward to it.

Thanks.

Nina

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Nina DiPrimio, PhD
Anderson Lab
Department of Bioengineering
UC Berkeley

Lab Phone- 510-664-4200

[Patrik D'haeseleer]

As a reminder, the wikipedia page on Aromatic compounds has tons of good starting points for biochemicals you might want to target, often with a link to a whole separate page for the compound:

http://en.wikipedia.org/wiki/Aroma_compound

One interesting exercise might be for each of you to pick your favorite scent from the table, and dig into the biosynthetic pathway for that compound.

For example, say you love the scent of roses - you'll notice that there are actually three compounds listed in the table for rose: geranyl acetate, geraniol, and nerol. You can dig into these a bit deeper to figure out which one is the key aroma compound in rose, but let's just go with geraniol, since it looks to be the simplest of those three. Here's the Wikipedia page for Geraniol, straight from following the link in the previous page:

http://en.wikipedia.org/wiki/Geraniol

Sometimes, this page will actually have a section on biosynthesis - this one doesn't. Sometimes, it may also have links to the compound (or even biosynthetic pathway) in KEGG or MetaCyc, which are the two main metabolic pathway databases. Again, this page doesn't, but it may be worth adding that information to the wikipedia page once you're done digging it up!

Since we don't have links to KEGG or MetaCyc directly, you'll have to look them up yourself. I prefer Metacyc, so go to this page and enter geraniol in the search bar:

http://metacyc.org/

You'll see three pathways, including geraniol and geranial biosynthesis (perfect!), geranyl acetate biosynthesis (one of the other compounds listed as smelling rosy!), and geraniol and nerol degradation (might contain other interesting aroma compounds as well). Let's check out that first pathway:

http://metacyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-5829

You'll notice that it actually starts out with geranyl diphosphate, coming from the geranyl diphosphate biosynthesis pathway. If you want to see the actual structures of the compounds, click on the "More Details" button.

Are any of these enzymes actually present in E. coli or yeast? Clicking on the "Species Comparison" button will give you E. coli by default (pathway not marked as present, and none of the enzymes are present). If you want to check S. cerevisiae as well, you can click "Change Organisms" at the bottom of the page.

Turns out neither E. coli nor S. cerevisiae have the key geraniol synthase enzyme to go from geranyl diphosphate (GPP) to geraniol, so you'll have to add that one at the very minimum. You could have guessed as much, because the pathway page says "Expected Taxonomic Range: Viridiplantae" (aka plants). Now, how about that geranyl diphosphate biosynthesis pathway?

http://metacyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-5122

With this one you're in luck: "Expected Taxonomic Range: Archaea , Bacteria , Eukaryota"! This single-enzyme pathway happens to be extremely common, and occurs both in E. coli and S. cerevisae (see also under "Some taxa known to possess this pathway include..."). IPP and GPP are also a precursors to a number of biofuels and other bioproducts, so you should be able to find some tricks in the literature on funneling more metabolic flux through those pathways.

Congratulations - you only need a single gene to produce geraniol in E. coli! Well, at least in theory anyway. Now you have to start worrying about whether you'll be able to express a plant enzyme in a bacteria, etc...

Patrik

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[Nina DiPrimio]

Thanks Patrik for following up with those links!