Transgenic yeast used to 'brew' lsd

[retired_chemist]

I don't have access to a university library anymore, so I cannot cite exact references but anyone who does can easily find that there are extenstive treatises on the vast amounts of research and effort that were put into simply refining the process by which claviceps and other ergot producing species would produce acceptable yields of ergotamine.

I'm not a biochemist, just someone who looked at the literature a long time ago. My distinct impression is that industrial production of ergotamine is quite an impressive accomplishment that has involved not only careful optimization of culture conditions but also numerous isolations of fungal strains over many generations, selected for maximum ergot production.

I think Joe Hippie would have a hard enough time reproducing the ergotamine production technology as it exists today. If he did, with a viable bio-source of basic ergotamine base there would not be much need to develop any new technology.

It's only the total synthesis of lysergic acid itself which is impractical. Synthesis of LSD from ergotamine is fairly straightforward.

PIHKAL, on the other hand is all very straightforward chemistry. Maybe you could create it all in a greenhouse, but it's still always going to be pretty easy to just do it in a lab. ;-)

 

[IGNVS]

this is exactly where i want to see things going. ive been thinking about stuff using that process for a whilllee now, do you know if anyone has actualy done it yet though?

 

[kidamnesiac]

^Click on the link in my post

Firstly, what class should I take for this stuff?

Secondly, would I be right in assuming this would be much easier with compounds such as a,O-DMS?

Biochemistry, molecular biology, organic chemistry, genetics, cellular biology, p-chem, systems biology, bioengineering for undergrad, then do your focus on it in grad school and read, read, read the literature.

And yea, a,O would be a good target, but again, the synthetic route is so damned easy, why bother?

r_c-unfortunately, a lot of the refining processes for improvement of batch culture, improved production ergotamine strains was done in industry (sandoz/whatever they are now) and hence not super available. MV Smith compares the culturing of claviceps strains to a slightly more advanced form of yogurt making, which it does seem on reading his formulas. Obviously the toxicity is important, and extraction of alkaloids also difficult. But this would not change with transgenic yeast or bacteria.

Also, PIHKAL is, for the most part, easy chemistry, you're absolutely right. It was a convenient example. How about an importation of the salvinorin operon, or the ibogamine genes? things with nasty synthetic routes and fragile skeletons.

Where I really see this going in real academia is towards marine natural products. There are so many interesting ones and it is so damned difficult to collect/culture those things, as well as the nasty synthetic routes that yield .001 grams of material in .02% yield, that it only makes sense to go this way.

F&B-If I remember the paper you're talking aboot, they did this with the amino acids on ergotamine right? And if I remember, it was only something like a methyl proline instead of regular proline on the 3AA position. driving eq. with a alkylated, 2nd amine instead of ammonia might be a lot more tricky, if not impossible with the native enzymes. I would think that N would have to come from an arg, but I'm not sure, again, missing my paper stash.

OK, this is funny, type in lysergic acid amide biosynthesis into google images, and F&B's and MGS avatars come up!

 

[vecktor]

All the key genes for ergoline biosynthesis have been isolated. it is now old technology to insert these and add a promotor for shake n bake biosynthesis in some suitable bacteria.
there was a PhD thesis from some american uni last year sometime that elucidated all the steps and the enzymes nvolved.
but it will remain cheaper to ferment C paspali for the moment, the technology is mature and reliable. and there is enough manufacturing capacity to supply the global demand for ergolines. and the manufacturing is concentrated in a few companies who understand c paspali fermentation.
the commercial strains high alkaloid strains of C paspali are available to any legitimate manufacturer that wants them.I can't see Sandoz or anyone else devoting much energy to GM production of Lysergic acid. there is perhaps an incentive to develop the biosynthesis of 2-methyl lysergic acid, as this is currently not possible even supplementing c paspali with 2 methyl tryptophan.

V

 

[hussness]

I think reengineering catabolic enzymes whose sequences are highly conserved (eg. the glycolytic system) would likely be a lot easier. Many of these tertiary and quaternary structures are fairly well understood; there is also a lot known about the kinetics and variablility of substrates in this area. I think an easier problem is to try and focus on a discrete set of chemical transformations where chirality is introduced or rearranged.
Theoretically, semipermeable membranes could be used to prevent or reduce the toxicity involved with having too much substrate and/or product.
And in case anyone gets any ideas, ergotism isn't something you want to mess around with.

 

[kidamnesiac]

I'd love to be proven wrong, but AFAIK and can see, the operon is not that straightforward. The dimethylallyltryptophan synthase gene, the first in the pathway, has been located, but it is directly upstream of the non-ribosomal peptide synthetase(s) (NRPS) genes, which would be the last step in the ergotamine synthesis pathway. It also looks like what genes are known are bi-directional, making control via a simple lac promoter not so easy. Most of the other enzymes, or genes, have not been explicitly identified. The only other one that seems to be certainly identified is the lysergol ->lysergic acid oxidizing p-450 based enzyme.

I would argue that un-natural product synthesis is not shake-and-bake. For instance, in this pathway, if your first enzyme is the DMAT to alkylate tryptophan, you have two problems. You need a way to upregulate tryptophan production because a bacterium makes it in minute quantities as it is something like .5% of AA in e coli (1.1% in all organisms). If you start stealing it all to make ergoloids, the bacterium is hurting and can't make it's normal proteins properly.

The second obvious problem is the lack of a strong source of dimethylallyl (pyro)phosphate. The non mevalonate pathway (methylerythritol phosphate pathway) in e coli is weak as there is not a huge need for DMAPP in eubacteria. However, what is there is important in things like membrane synthesis and upkeep and protein prenylation. Steal your isoprenes and your bug could be seriously hurting.

Then there are the generic factors of having soluble transgenic protein and them not being toxic to the cell, making sure enzymes can fold in a new organism (the number of unique chaperones is amazing and assuming having a proper but non-unique one in your bacteria is a poor assumption to make), proper regulation of things like phosphorylation, prenylation, alkylation, etc, making sure you have sufficient levels of cofactors, making sure your cofactors (like B12) don't become inactivated by substrates and need reactivases (I have one of these now), compartmentalization/substrate channeling importance (some intermediates need to be acted on with teh quickness), feedback inhibition by compounds normally found in e coli but not the native organism, and most importantly, especially when dealing with higher organism proteins in lower organisms, post translational modifications! AND these things all have to take place in a suitable robust bug for alkaloid production that is lacking in non-specific proteases. It's easy to get the former and the latter individually, but together is not nearly as common.

It's not shake and bake or people wouldn't be getting absurd amounts of money and 3 years of nature pubs, even if it is a health related endeavor.

I would like to see that thesis though, can't find it on scholar or pubmed.

 

[kidamnesiac]

Theoretically, semipermeable membranes could be used to prevent or reduce the toxicity involved with having too much substrate and/or product.

But what do you make it permeable to? It would have to be only the final product and if any intermediate is toxic your stuck. Now you need to engineer either a transporter protein or membrane protein to be permable to your final product only. I would not want to be that grad student A generically semi-permeable membrane isn't really feasible in batch culture, it would be like having your stomach full of holes.

 

[tobala]

I'm in over my head here, but how amenable are chiral centers to enzymatic manipulations?

 

[Beenhead]

Wow, this is pretty cool, its actually quite similar to waht I do at work every day. And pretty much all of the equiptment as well... Now all i need is some Fungus DNA...

 

[hussness]

Yeah, making lsd at work is a good way to lose your job...and gain a criminal record...I'm sure you're being facetious about this though.

 

[kidamnesiac]

It is, however, a great way to extend your chromatography skills as well as spend a rainy weekend

no, i haven't made lsd

 

[tobala]

^^^^ Easy lad. I think he works at a brewery...

 

[kidamnesiac]

as far as chiral centers, many synthetic chemists are starting to look at enzymatic reactions in industrial preps because of the inherent chirality. enzymes are inherently chiral so many reactions they catalyze will work with absolute stereospecificity.

however, you probably wont be able to make d from l. but a chiral enzyme is the reason that all natural ephedrine is the d form.

 

[Beenhead]

It is, however, a great way to extend your chromatography skills as well as spend a rainy weekend

no, i haven't made lsd

I looove chromatography!

 

[Ham-milton]

I'd imagine that splicing the LSA-producing gene into something else would be rather simple, but trying to get a plant to produce an entirely synthetic drug? That's gotta be way more difficult.

I'd settle on making a transgenic yeast that'd pump out THC; That wouldn't be nearly as difficult.

 

[tobala]

^^^^ Easier to get the botanical organism to do it for you...

 

[kidamnesiac]

and would you really want to smoke yeast?

the extraction, while easy, just makes it one more step for joe hippie. but yea, you could manifest gallons of honey oil w/teh quickness that way.

again, the terpenoid pathway would need to be imported and tweaked.

 

[Enix150]

I'd imagine that splicing the LSA-producing gene into something else would be rather simple, but trying to get a plant to produce an entirely synthetic drug? That's gotta be way more difficult.

I'd settle on making a transgenic yeast that'd pump out THC; That wouldn't be nearly as difficult.

Old thread, but I thought I'd post some updates!

Who needs weed when we've got THC-producing yeast?

Researchers from the Technical University of Dortmund in Germany have reportedly discovered a way to genetically engineer yeast to produce both THC and cannabidiol. THC is the primary psychoactive chemical in cannabis which cannabidiol (or CBD) is a potent anti-inflammatory compound. Synthetic THC is already sold under brand names like Cesamet. It's often used to treat nausea associated with HIV or cancer therapies. However getting yeast to produce just those chemicals, rather than having to strip them from the plants themselves, could greatly reduce the cost of their production compared to conventional chemical synthesis.

Their study, published in the journal Biotechnology Letters, only covers the THC-producing yeast strain -- information on the cannabidiol strain remains unpublished. Unfortunately, these strains only produce limited quantities of their respective compounds and require a sugar-based precursor to do so. The team hopes to eventually replicate the full THC-production pathway and has already joined forces with THC Pharm of Frankfurt to scale up production.

Harvard scientists to make LSD factory from microbes

Around 20 tonnes of lysergic acid, a precursor of LSD, are made each year and turned into real medicines, such as nicergoline, a treatment for dementia. The drug is purified from big vats of fungus (which make the compound naturally) using technology developed decades ago.

With the tools of synthetic biology, Wintermute thought they might do better. The ergot fungus takes lysergic acid and turns it into a huge variety of exotic molecules. They could mix and match biological pathways from different species of ergot fungus and make potentially new drug molecules. They might even come up with a next generation dementia drug.

Wintermute gave an update on the project last week at the Synthetic Biology 5 conference at Stanford University. As yet he is not making any lysergic acid, but he has dropped two of the six required biological pathways into baker's yeast, which merrily churns out intermediate compounds. At a rough estimate, he expects a litre of modified yeast in broth will produce a gram of lysergic acid. Once made inside the yeast, the drug should pass through the cell walls, where it can be separated out and purified.

This could be the first step towards a new living factory for making LSD, and thanks to previous experience with microbes, scaling up this kind of technology should not be too arduous for the pharmaceutical industry. The work brings to mind a more developed effort by Jay Keasling and others to engineer microbes to churn out useful biofuel components.

 

[SONN]

IMO this raises questions about the whole kykeon argument and whether or not there were strains of ergot being brewed into beer with lysergic repercussions in the olde days

 

[serotonin2A]

IMO this raises questions about the whole kykeon argument and whether or not there were strains of ergot being brewed into beer with lysergic repercussions in the olde days

I'm not really sure why you think this raises questions. The Greeks lacked the technology to culture ergot. It isn't as simple as brewing beer. They didn't have any concept of sterile technique.

If you think that they were genetically engineering yeast to express the enzymes responsible for synthesizing lysergic acid amides then you are overlooking the fact that the required technology is less than 100 years old.