Molecular Cloning and the Synthesis of Recreational Drugs

[Riemann Zeta]

Novel proteins, though, is not the same either. By novel, you means proteins that hadn't been seen before or proteins that weren't produced naturally before?

By novel, I mean designing and engineering your own proteins to catalyze target synthetic reactions. For example, taking the lysergyl peptide synthetase proteins and the enzyme ergometrine synthetase from the amazing (but nasty) fungus and modifying them to make your own favorite ergoloid. In essence, rolling your own "diethyllysergamide synthase" protein(s) and employing it/them in vitro, with the lysgeramide moiety attached to whatever kind of grabby chromatography beads/polymer you want. Make it on the substrate, unlink & elute, purify out as the freebase in an organic solvent and finally recrystalize the salt.

Obviously, it is going to take a really fucking long time before the technological foundation necessary to pull something like this off is developed. But it's still cool to think about [cue voice] "The World Of Tomorrow." Those lucky futuristic bastards with their robots and their endless supplies of LSD.

 

[kidamnesiac]

Theseeker-I'm not sure when you did your PhD, but I have the feelings things have changed a little since then. I've expressed a dozen or so proteins from a number of organisms and purified them, some in volumes and purities for NMR and crystallography concentrations. I see people in the lab I spend my days in expressing eukaryotic enzymes and proteins every day from our only source, e. coli. There are now plasmids that can be co-transformed that contain all of the genes and tRNA for rare codon insertion, chaperones, glycosylation, etc that allow people to express a lot of proteins. If that doesn't work, yeast is also a great option which seems like the best option for those lysergide making enzymes, eh? Are the proteins most groups study the ones amenable to easy expression and purification? sure. But I bet a lot of the proteins you worked on were the bitches that were really stubborn for everyone else. I also agree with you about expressing e. coli proteins in e. coli, the hardest protein I ever tried to purify was a coli native heterodimer. This isn't called heterologous expression though, it's homologous!

Purification of small molecules from bacteria is beyond banal compared to some of the extractions from plants or marine organisms for those whacky rare molecules used in cancer research. Even taxol is a bear to purify. Even better, the I believe the Keasling group was able to get their yeast to export the artemisinin from the cells. Spin down the cells, acid/base the supernatent, run a column, proceed. And yes, in MOST cases it will probably just be easier to get the products from their natural source. But this is true of most synthetic approaches to, see morphine, THC, lysergic acid, and thousands of other non-drug-like semi-complex molecules. So there are some economic and sociological reasons to express these things in bacteria, like not having to worry about growing poppies in big fields readily accessible to young hooligans who frequent this forum.

@Riemann-why in ergots name would you want to put the non-ribosomal peptide synthetases into the bacteria if all you want is the lysergic acid or simple amide from it? Those NRPS's are insanely complicated (and freaking awesome) but that would be such a pain to get right for the reasons I explained before. But yes, part of my dreams a few years ago were to get these enzymes together and then do some reverse engineering to make lysergamides. It is possible, but I don't think it will be bathtub accessible because you would likely still have to purify the molecules to some degree (unless you want to just drink some bacteria broth).

At any rate just a month or two ago someone posted a nice review from nature chemical biology about exactly this type of stuff including the isoquinolines like morphine and complex tryptamines. So some people are getting there. But they are not working on making LSD, so it's up to you guys. I will try to remember to post some other just stinking awesome papers about doing the type of work on antibiotics of high complexity and medium size. Expression of the enzymes, reverse engineering and evolution of the synthesizing enzymes, and purification of the products.

 

[LabRatNW]

Check this out: Researchers prove custom-designed enzymes are possible

Sweet, right?

 

[Rectify]

Everything is possible, but I don't think researchers have designed functional designer polypeptide enzymes yet.

I'll stick with small molecules.

 

[theseeker]

@kidamnesiac- excellent reply. Point well taken- ie. not growing mass poppy field etc. Didn't think of that. Just defended my PhD last October but ONLY worked on two artificial proteins. They did indeed suck but got them to express!!! I guess this experience slanted me and made me turn a jaundiced eye towards protein purification from artificial genes- lost all my hair (hahaha). It still haunts me. Thanks for educating me. My latest response was based on:

Graslund, S., et al., Protein production and purification. Nat Methods, 2008. 5(2): p. 135-46

 

[kidamnesiac]

From the Daptomycin wiki:

The molecular engineering of Daptomycin, the only marketed acidic lipopeptide antibiotic up to date (Figure 8), has seen many advances since its inception into clinical medicine in 2003.[15] It is an attractive target for combinatorial biosynthesis for many reasons: second generation derivatives are currently in the clinic for development;[16] Streptomyces roseosporus, the producer organism of daptomycin, is amenable to genetic manipulation;[17] the daptomycin biosynthetic gene cluster has been cloned, sequenced and expressed in a S. lividans;[16] the lipopeptide biosynthetic machinery has the potential to be interrupted by variations of natural precursors, as well as precursor-directed biosynthesis, gene deletion, genetic exchange, and module exchange;[17] the molecular engineering tools have been developed to facilitate the expression of the three individual NRPS genes from three different sites in the chromosome, using ermEp* for expression of two genes from ectopic loci;[18] other lipopeptide gene clusters, both related and unrelated to daptomycin, have been cloned and sequenced,[8] thus providing genes and modules to allow the generation of hybrid molecules;[17] derivatives can be afforded via chemoenzymatic synthesis;[19] and lastly, efforts in medicinal chemistry are able to further modify these products of molecular engineering.[16]

I can provide those papers for anyone if they are interested, they are facinating.

Everything is possible, but I don't think researchers have designed functional designer polypeptide enzymes yet.

I'll stick with small molecules.

So aside from what has been done with daptomycin, designer terpene synthetases have been made too:

Y. Yoshikuni, T. E. Ferrin, and J. D. Keasling. 2006. “Designed divergent evolution of enzyme function.” Nature 440:1078-1082.

Or how about the evolution of an enzyme to make a lipitor precursor?

Fox et al. "Improving catalytic function by ProSAR-driven enzyme evolution." Nature Biotech 25 338-344

You want de-novo enzyme design?

Röthlisberger, D et al. "Kemp elimination catalysts by computational enzyme design." Nature 453, 190-5.

Jiang L et al. De novo computational design of retro-aldol enzymes. Science 319, 1387-91.

The last two there are available for free from the baker group (UW) website. These are the creme de la creme, but there are many more examples.

@seeker-i hear you about the hair loss! and that paper is interesting (just skimmed it, but saw the 30% remark). I would say while 30% can be purified, this is not especially relevant to recombinant production of small molecules. There does not need to be high expression and hence concentrations (which is the cause of most problems in purification I think, things like inclusion bodies and toxicity), nor does the enzyme need to be stable through a purification. They just need to work in concert in relatively low concentrations inside the cell, so the hurdles are still high, but not insurmountable.

 

[mannequin]

supposedly the nazis were doing it with lots of drugs.. mainly heroin

 

[LabRatNW]

Everything is possible, but I don't think researchers have designed functional designer polypeptide enzymes yet.

I'll stick with small molecules.

That's what I'm talking about. How about an enzyme set that produces LSD as the final product? Or 2C-E? or Foxy?

 

[Riemann Zeta]

I see people in the lab I spend my days in expressing eukaryotic enzymes and proteins every day from our only source, e. coli. There are now plasmids that can be co-transformed that contain all of the genes and tRNA for rare codon insertion, chaperones, glycosylation, etc that allow people to express a lot of proteins.

Kidamnesiac: now you have piqued my curiosity. How does one achieve proper glycosylation of a given protein of interest in a transformed prokaryotic expression system? Only a handful of simple glycosylated proteins have ever been detected or produced in prokarotic cells. If you or the individuals in your lab have a method for producing a given glycoprotein in bacteria, I'd get to the nearest patent office as soon as possible, because people would pay a shit-ton of money for this ability.

why in ergots name would you want to put the non-ribosomal peptide synthetases into the bacteria if all you want is the lysergic acid or simple amide from it? Those NRPS's are insanely complicated (and freaking awesome) but that would be such a pain to get right for the reasons I explained before. But yes, part of my dreams a few years ago were to get these enzymes together and then do some reverse engineering to make lysergamides. It is possible, but I don't think it will be bathtub accessible because you would likely still have to purify the molecules to some degree (unless you want to just drink some bacteria broth).

Bathtub accessible? Give me a little credit here--I'm not talking about a couple of tweakers cooking up gutter meth. Obviously, even getting lysergamide synthetase and ergometrine synthetase to express in bacteria such that they might be harvested and purified for synthetic (cell-free) reagent purposes is a logistical nightmare that makes any such approach ridiculously impractical with contemporary technology. It's just fun to think about .

 

[kidamnesiac]

Here is one example I thought of, there are others, but I only work with prokaryotic enzymes anyway, to hell with the complicated stuff I say.

N-Linked Glycosylation in Campylobacter jejuni and Its Functional Transfer into E. coli

N-linked protein glycosylation is the most abundant posttranslation modification of secretory proteins in eukaryotes. A wide range of functions are attributed to glycan structures covalently linked to asparagine residues within the asparagine-X-serine/threonine consensus sequence (Asn-Xaa-Ser/Thr). We found an N-linked glycosylation system in the bacterium Campylobacter jejuni and demonstrate that a functional N-linked glycosylation pathway could be transferred into Escherichia coli. Although the bacterial N-glycan differs structurally from its eukaryotic counterparts, the cloning of a universal N-linked glycosylation cassette in E. coli opens up the possibility of engineering permutations of recombinant glycan structures for research and industrial applications.

Science 2002

Thinking about it though, it should be pretty unimportant for most applications of in-vitro molecule manufacture. The bacteria don't give a shit about localization or complex glycosyl signaling, only if the PTM are relevant to things like making substrate channeling complexes or activation would it be needed.

I'm now vacillating on the bathtub statement. Since DMAPP genes have been inserted and are stable via the Keasling group, all you need to feed is high tryptophan and then oh...8 enzymes, likely part of an operon, to be expressed in your bug and you get Lysergic acid. One more good one to lysergide. harvest cells, (lyse if they don't actively pump out the product, collect supernatent) gentle acid/base, quick crystallization. Doesn't seem outside the realm of the typical local mdma maker. Easy as growing mushrooms in the closet? No, but no harder than being a full time ganja grower either.