Increasing production of compounds already present in a plant?

[Andrew Willoughby]

How would you get a plant to produce more of a compound? Increasing the amount of caffeine in coffee, antioxidant anthocyanins in berries, or even that cyanide precursor in apple seeds?

I'm assuming that it depends on how the chemicals are produced in the plant of course. When I googled caffeine biosynthesis for instance, I saw that it involves several different steps. I'm guessing if you're using genetic engineering techniques it'll be hard to figure out exactly where your production is bottling up, but still the idea would be to increase the amounts of the enzymes needed in caffeine's sake at least to produce theobromine and turn it into caffeine. And I thought that you could increase the amounts of the enzymes (N-methyltranferases I think is what i read and add theobromine to the plant and let it turn that into theobromine into caffeine, but that's lazy! :D. How do you get an organism to produce more of an enzyme? Or I guess more completely, I don't want to know how to add a new biosynthetic pathway, but to increase the efficiency of one already present. I guess if it's too specific to the pathway in question, then stick with caffeine. Anthocyanins sound too tricky from Wikipedia (several enzymes at once) and I'm not interested in poisoning anyone so apples won't be much use.

Also slightly unrelated question but I don't really want to be making two posts and it's in the same vein, genetic engineering and compounds that plants produce, but if a plant produces a chemical in only its roots, or its leaves or its fruits, how would you get it to produce that compound in other parts of the plant? Is that a function of epigenetics? I'm thinking of things plants produce like insecticides and aromatic compounds.

[Dakota Hamill]

There isn't an easy answer to your questions.  If you can figure out an easy answer, congratulations you have a billion dollar company.  Someone could spend an entire PhD thesis on figuring out how to optimize one single biosynthesis pathway for one end product, and people do.  There are companies that spring up with millions in funding that seek to make bio-synthetic pathways economically/commercially feasible for the production of certain small molecules.  It takes a lot of tweaking.  You have to consider many things.

1) the metabolic demand on the cell to produce your end product, as well as its toxicity (if any)

2)codon optimization for the enzymes necessary for each step of your reaction (rare codons can decrease how much of the necessary enzymes are made)

3) side-reactions which will "steal" your substrate or its intermediates along the way to the desired final product

4) expression of your desired enzymes in correct ratios (ie - you could put the different enzymes under the control of well characterized promoter and RBS with varying degrees of expression).  Some enzymatic reactions could be a bottleneck, so if you could pinpoint that, and increase the expression level selectively of that one enzyme, you could increase the efficiency of your pathway. 

5) Is all the work you put going to be worth it?

As an example, caffeine is probably a poor target molecule, but I understand you are probably asking to use it as an example.   So much of it is already harvested from coffee beans that it'd be a waste of resources to try to produce it in another organism.

A few companies try to do things like which you are speaking, I'm sure there are many more but these are just a few names I remember

http://www.metabolix.com/

http://ginkgobioworks.com/

http://www.bio-amber.com/products/en/products/succinic_acid   although I think they might just isolate it from agro waste products

I'm sure the caffeine biosynthesis pathway is well documented, so read journal articles about it to familiarize yourself with some of the genetic engineering tools they use, and then, apply it to a compound with a commercial value that is expensive to produce synthetically and could be an ideal target for production recombinantly.   1,000,000,000x easier said than done.

Also your idea of "feeding" an intermediate to an organism over-expressing a necessary enzyme to catalyze a final step to a finished product isn't lazy, it's a good idea.  People do it.  If there is a complex reaction that an enzyme can do well, which synthetic organic chemists can't match, sometimes its commercially viable to feed the organism an intermediate and let it spit out the finished product.

If you're dead-set on plants, metabolix is probably the closest thing you're looking for since they use plants.

There was another company that uses plant tissue culture to make Taxol intermediates but I forget the name of it. 

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[Matt Lawes]

Dakota has made a very nice summary of the main considerations. Admirably brief while being technically correct! Very nice job!

Andrew your original question is excellent and the field of metabolic engineering - whether improving endogenous production or transgenic production is very complex technically. But also a huge opportunity both scientifically and commercially.

>matt

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Dakota Hamill <dko...@gmail.com> wrote:

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[Andrew Willoughby]

Sadly maybe not a lot of commercial potential besides novelty, but an idea I've been playing around with is caffeinated oranges. Not to harvest the caffeine itself, but just as an alternative to coffee in the morning. I knew that citrus plants already have slight amounts of caffeine in them (very VERY small amounts), so even if the synthesis pathway is different from coffee's it doesn't seem as hard to increase the caffeine amount as it would be if you had to completely engineer the pathway into the plant. The problem is I have found about 8 articles that would help, but they are so expensive! 

[Cathal (phone)]

Don't ever pay for articles. Look on libgen.org, or ask for PDFS in IRC or mailing lists. Or twitter with the #paperfairy hashtag.

Also, if you don't mind your name/location/work being public, install and use the "open access button" in your browser to help demonstrate where closed access harms science.

On 22 June 2014 05:23:27 GMT+01:00, Andrew Willoughby <andrew.wil...@gmail.com> wrote:

Sadly maybe not a lot of commercial potential besides novelty, but an idea I've been playing around with is caffeinated oranges. Not to harvest the caffeine itself, but just as an alternative to coffee in the morning. I knew that citrus plants already have slight amounts of caffeine in them (very VERY small amounts), so even if the synthesis pathway is different from coffee's it doesn't seem as hard to increase the caffeine amount as it would be if you had to completely engineer the pathway into the plant. The problem is I have found about 8 articles that would help, but they are so expensive! 

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[Dakota Hamill]

Your comment about oranges made me think of the Munich iGEM team that made yeast expressing the synthesis pathway you are speaking about for caffeine, as well as for a few other interesting compounds.

http://2012.igem.org/Team:TU_Munich/Project/Caffeine

Though I havn't read their work in detail, iGEM teams usually have pretty detailed pages describing their thought process and design of the specific gene circuit.

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[John Griessen]

On 06/21/2014 11:23 PM, Andrew Willoughby wrote:
> even if the synthesis pathway is different from coffee's it doesn't seem as hard to increase the caffeine amount as it would be if
> you had to completely engineer the pathway into the plant.

Excellent lateral thinking! Ideas like this will flower even in an environment
of legal barriers, costs of research that are normally too high, and technical difficulty.
Go DIYBIO.

[Mega [Andreas Stuermer]]

mutation breeding. It's not safe, but it's totally legal

[Cathal (phone)]

Who says it's not safe? :) Certainly less safe than GM, but..

On 22 June 2014 18:29:35 GMT+01:00, "Mega [Andreas Stuermer]" <masters...@gmail.com> wrote:

mutation breeding. It's not safe, but it's totally legal

[Matt Lawes]

Yes there would be no crop improvements without mutation breeding and selective breeding. DIYBIO is still science. Luddites and envirofascist propagandistic agendas are at odds with the spirit of open scientific endeavor.

In my opinion .... I should add.

>matt

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"Cathal (phone)" <cathal...@cathalgarvey.me> wrote:

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[Sebastian Cocioba]

if you need help or want some tips from a technical standpoint, feel free to ask. I've been messing with plants using most of the routine methods of plant transformations for a few years now and feel comfortable enough to be able to throw in my two cents. Plant transformation is a fairly simple procedure and can be done with not too much overhead. 

The main roadblock seems to be getting a hold of agrobacterium and a functional integration vector. Universities may donate but local, state, federal, national restrictions may apply.

I'm still working, albeit slowly, on my gene guns and will offer more on that as I reach the necessary milestones.

Protoplast transformations and regeneration is more expensive and very skill dependent. Trial and error seems to be the best way to debug protoplast issues. The cell wall degrading enzyme cocktails are expensive. Working on alternative sources as well as a possible recombinant route for said enzymes.

All and all the technical side is very straight forward. The genetic side will, of course, be much trickier. Fishing out whole pathways and expressing that in plants is still a difficult undertaking. A genetic circuit that just makes all the parts all the time does not always lead to increased yield. Fine tuning may be needed. One could start by trying to compare the pathways between orange and coffee/cacao/tea pathways as see where they start to differ and just add the enzymes constitutively to express the missing or different parts. Not sure it will be effective but could also get your hands dirty with plant transforms if time isnt a factor. Primers are fairly cheap (Invitrogen Value Oligos), cloning can get pricey but im sure there are biobricks that can lend a hand. Once you have your circuit, slap on some homologous flanks like the ones used in typical chloroplast transformations like the trnA (alenine) and trnI(isoleucine) intergenic regions and then shoot it into a plant via gene gun or protoplast PEG dna uptake and let the miracle of homologous recombination do all the work. Again, technical side is an endeavor but doable. 

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[Andrew Willoughby]

Thanks! Right now this isn't anything more than a thought experiment. But if in college I have any autonomy in a lab, I'll definitely ask for help. I have managed to download a few books on tissue culture. Reading and researching is as far as I can take this until college starts. (I'm a recent high school grad)

[Dakota Hamill]

You have plenty of time to learn then and get into a lab to do some hands on stuff.  If your school has an iGEM team, try to join that, and if it doesn't, and you're really driven, try to convince them to make one once you get to know your professors better and observe who might make a good adviser.

On Mon, Jun 23, 2014 at 1:39 PM, Andrew Willoughby <andrew.wil...@gmail.com> wrote:

Thanks! Right now this isn't anything more than a thought experiment. But if in college I have any autonomy in a lab, I'll definitely ask for help. I have managed to download a few books on tissue culture. Reading and researching is as far as I can take this until college starts. (I'm a recent high school grad)

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[Andrew Willoughby]

My school used to have an iGEM team but hasn't the last two years. I'm hoping to start it back up!

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[Andrew Willoughby]

Oh nevermind it looks like they joined 2014!