ABS biosynthesis

Lego, rumour has it, wants to biosynthesise acrylonitrile, butadiene styrene (ABS), the resin that gives their blocks their firm hold and transgenerational lifespan. This is cool for three reasons:

1. metabolic engineering is cool by definition,
2. Lego is cool by definition and
3. one or two steps link back to a cool gene I found in Geobacillus

Chemistry

So what might they do to biosynthesise their resin? The processes are rather straightforward and one has to go out of one's way to dream up a cool route. In fact, there is a lot of repetition.

The three monomers for the polymerisation are styrene, acrylonitrile and butanediene. These would be made separately. But there are several commonalities, such as the terminal ene group.

There are a few ways to get a terminal ene group:

1. Have a 2,3-ene and tautomerise it
2. Have a 2,3-ene and terminal carboxyl and eliminate the carboxyl
3. Reversible dehydration
4. Irreversible dehydration via phopharylated intermediate
5. Oxidative decarboxylation (oleT encoded p450-dependent fatty acid decarboxylase from Jeotgalicoccus sp.)

My guess is that their major challenge is that they will have to extensively modify a few enzymes and will be plagued with detection and screening. Nevertheless, I am still going to talk about the chemistry as it is a good excuse to sneak in a cool set of genes from Geobacillus.

Styrene

There are two way to biosynthesise styrene. The simplest is decarboxylating cinnamic acid, while the more interesting one by dehydrating phenylethanol.

The tourist route

Phenylethanol —also unglily called phenylethyl alcohol— is in turn made from phenylacetate, which is made from phenylpyruvate.

Recently, while analysing a transcriptomic dataset for Prof. D. Leak, which resulted in an awesome website, www.geobacillus.com, I stumbled across a really cool enzyme encoded among phenylalanine degradation genes, that I speculate is a phenylpyruvate dehydrogenase. This is a homologue of pyruvate dehydrogenase and follows the same mechanism, namely a decarboxylative oxidation followed by CoA attack.

There are other ways to make phenylacetate, but none allow such a shameless plug for my site —in fact, I should have talked about the 2-phenylethylamine biosynthetic route instead.

In nature the phenylacetate will go down the phenylacetate degradation pathway (paa genes), but it could be forced to go backwards and twice reduce the carboxyl group. Phenylacetaldehyde dehydrogenase is a common enzyme, which even E. coli has (faeB), but the phenylethanol dehydrogenase is not. I found no evidence that anyone has characterised one, but I am fairly certain that Gthg02251 in Geobacillus thermoglucosidasius is one as it is an alcohol dehydrogenase guiltily encoded next to faeB, which in turn is not with phenylethylamine deaminase (tynA).

So, that is how one makes phenylethanol. The dehydration part is problematic. A dehydratase would be reversible, but offers the cool advantage that it can be evolved by selecting for better variants that allow a bug with the paa genes and all these genes to survive on styrene as a carbon source. The alternative is phosphorylation and then dehydration as happens with several irreversible metabolic steps.

The actual route

That is the interesting way of doing it. Whereas the simple way is rather stereotypical. In plants there are really few secondary metabolites that are not derived from polyketides, isoprenoid, cinnamate/cumarate or a combination of these. Cinnamic acid is deaminated phenylalanine via a curious elimination reaction (catalysed by PAL). In the post metabolic engineering breaking bad I discuss how nature makes ephedrine, which is really complex and ungainly and then suggest a quicker way. Here the cinnamic acid route is actually way quicker as a simple decarboxylation does the trick. S. cerevisiae to defend itself from cinnamic acid, it has an enzyme PAD1p that decarboxylates cinnamic acid. Thefore, all that is needed is PAL and PAD1.

butanediene

Previously I listed the possible routes to an terminal alkene, which were: 

1. Tautomerise a 2,3-ene
2. Decarboxylate a 2,3-ene with terminal carboxyl
3. Dehydrate reversibly
4. Dehydrate irreversible via phopharylated intermediate
5. Decarboxylate oxidatively

In the case of butanediene, it is a 4 carbon molecule already, which forces one's hand in route choice. Aminoadipate is used to make lysine when diaminopimelate and dihydropicolinate are not needed. That means that a similar trick to the styrene biosynthetic route could be taken, namely aminoadipate is eliminated of the amine by a PAL mutant, decarboxylated by a PAD1 mutant and then oxidatively decarboxylated by a mutant OleT. But that requires changing a lot the substrate for three steps and the cells went to a lot of effort to make aminoadipate, so it is rather wasteful route.

Another way is to co-opt the butanol biosynthetic pathway to make butenol and dehydrate that.

A better way is to twice dehydrate butanediol.

As mentioned for styrene, a reversible dehydration means that selection could be done backwards. However, pushing the reaction to that route would require product clearance, otherwise there will be as much alcohol as the alkene. With butanediol and butanol there is a production and a degradation pathway, which would mean that selection could be done with the degradation route, while the actual production with the production route.

acrylonitrile

That is a curious molecule to biosynthesise. There are nitrile degrading bacteria and some pathways make it, so it is not wholly alien. preQ0 in queuosine is the first I encountered. QueC performs a ATP powered reaction where a carboxyl is converted to a nitrile. I am not sure why, but a cyano group seems (=Google) less susceptible to hydrolysis than a ketimine for some reason  —methylcyanoacrylate (superglue) follows a different reaction. Beta-alanine could be the starting compound, but it would require so many steps that it is a bad idea.

Substituting carboxyl for nitrile (nitrilating?) on acrylic acid with a QueC like enzyme would be better. Acrylic acid is small so it can be made by dehydration of lactic acid, oxidative decarboxylation of succinate or decarboxylation of fumarate. The latter sounds like the easiest solution as there are many decarboxylases that use similar molecules, such as malate or tartrate decarboxylase.

Challenges

Basically, even if it seems like a crazy idea at first, the processes are rather straightforward —one or two engineered enzyme for each pathway—, but the chemistry is pretty hardcore, so the few engineered enzymes will have to be substantially altered. Given that the compounds are small, quantifying yields will be their main challenge. How one goes about designing a selection systems for these is an even bigger challenge as evolving repressors to respond to small and solely hydrophobic compounds would be nearly impossible... So they will have to do this most likely by rational design alone, which makes it seem like a crazy idea after all.

Metabolic engineering Breaking Bad

Disclaimer: Obviously, this is a speculative what-if out of intellectual curiosity and by no means condones narcotics and their creation.

The logo of Breaking Bad had it been bio.

Half the issues in Breaking Bad could have been solved if they had been using biocatalysis and metabolic engineering. The catch is that nobody has made a production strain and it is not a simple task.

Biocatalysis: greener and safer

Some time soon, biocatalysis and metabolic engineering will replace many heterocatalytic processes as it is more efficient, cheaper, safer and greener.
Worldwide, there is a big problem of exploding methamphetamine labs. This problem could be fixed by switching from toxic and dangerous heterocatalysis processes to green and safe biocatalysis ones.

Requirements

So if Walt and Jessie wanted to win the green chemistry award, what would they need to do?
Their major problem is the starting material and the production steps. So the whole lot. Therefore, they need to do metabolic engineering.
The major issue is that methamphetamine is not a natural compound, so extensive engineering would be needed to produce the final steps. However, once they laboriously made a production strain, they would need to set up a large-scale bioreaction, i.e. a brewing tank, extract the product by phase-separation, remove the solvent and purify by crystallisation as they normally do. Then the only worry then is that they may be as contaminant-prone as Hank is at brewing.

Starting point

Methamphetamine is a compound that looks like phenyalanine, but on the chiral α-carbon there is methyl group instead of a carboxyl one and the amine group is methylated.
The natural molecules most similar to methaphetamines are pseudoephedrine and ephedrine. These two diastereomers differ from the former in having a hydroxyl group (in different chiral orientations) on the carbon adjacent to the benzene ring. The biosynthetic pathway is known (PMID 22502775), but requires eleven steps, which have not been assembled exogenously in an orderly way.
Additionally, to convert ephedrine to methamphetamine new enzymes need to be engineered for the hydroxyl reduction, which is problematic. Consequently, ephedrine biosynthesis might not be the best route and instead something more radical may be in order.

Ephedrine biosynthesis

Ephedrine gets its name from the genus Ephedra, whose members produce it. Unfortunately the selective pressures for plant secondary metabolism are rather unusual (cool) and as a result the metabolic routes get a tad tortuous.
If one were to forget how plants like to do things, one would guess a simple pathway with a similar logic to threonine biosynthesis be present. Namely, the carboxyl is twice reduced and the remaining hydroxyl is isomerised to the right place.

The final step (not pictured), the N-methylation, would be simply accomplished by a SAM-dependent methyltransferase and is the only part that is correct.
In the isomeration step, one might anticipate that an enamine-ketimine tautomerism followed by an attack by water might occur ruining the effort. However, the isomerisation of homserine to threonine is done via a PLP enzyme which holds the amine, so this isn't the problem.

The problem is plants like to make second metabolism in an OCD way, starting from specific compounds (e.g. geranyl-PP, farnesyl-PP, cinnamoyl-CoA, coumaroyl-CoA, malonyl-CoA and acetyl-CoA), preferably using decarboxylative condensation.
The ephedrine pathway is no exception and shares the beginning of the pathway with many other compounds Phenylanine > cinnamic acid > cinnaomyl-CoA>>benzoyl-CoA. Then the unique part is that the benzoyl-CoA is condensed with pyruvate, reduced and transaminated.
In reality, whereas the full pathway is known, the genes themselves are not, simply because nobody has sequenced E. sinica. Although a group in 2009 has gone to the effort of making yeast take up the DNA of E. glauca via ion implantation —no kidding around there!— and make ephedrine, but they did not sequence a few hybrids or similar, but instead did a lot of tedious work with primers (PMID: 19280123). Consequently, their experiment would need to be repeated, but with sequencing.
Once the various genes are cloned into E. coli, preferable into a strain that overproduces phenylalanine (eg. from PMID: 17880710), the pathway would be optimised, giving an ephedrine-producing strain.

The last step

The last step is the trickiest.
After those few years of work are done, the hydroxyl needs to be removed. Biochemically, hydroxyl groups are normally removed in two steps, the hydroxyl group is removed without adding an electron pair to the molecule by a hydroxylase, therefore leaving a double-bonded carbon, which is then reduced by a reductase. In some rare cases, the hydroxyl is reduced away. The most famous example is ribonucleotide reductase. The mechanism is rather mental and ugly.

The dehydration route is a problematic option however. A modified 3-hydroxyacyl-ACP dehydrase and the enoyl-ACP reductase from the fatty acid biosynthetic pathway seem like good candidates. However, the dehydrated enolamine would spontaneously tautomerise and hydrolyse as mentioned above.
This might not be that catastrophic as the product would be phenylpropanone, which being similar to phenylpropane-dione, the product of the pyruvate-benzoyl-CoA condensation. It might be promiscuously transaminated again by the cathionine transaminase or by phenylamine transaminase.
Nevertheless it is an odd way of doing things.
The N-methylation must be done last as the product is slippery being so hydrophobic. Normally, biochemistry likes to put a handle to hold stuff like that, such as phosphates, CoA and glycosides. In this case, a N-glycosilation would be a good option. The best bet, however, would be to move the N-methylation step after the hydroxyl reduction, the methyltransferase cannot discern between the precursor for ephedrine or pseudoephedrine, so it is probably fairly accommodating towards amphetamine.

Crazy way

The carboxyl group needs to be replaced with a methyl group. This is not an option from a biochemists' perspective as C-C bonds cannot be made that easily, unless by condensation or transmethylation on aromatic structures. In a typical methyltransferase the methyl donor is SAM, while the acceptor is a nucleophile (Lewis base), such as an amine or a hydroxyl that has been deprotonated by a catalytic acid. From a technical point of view as far as I can tell there should not be anything forbidding a PLP and SAM dependent decarboxylative C-methylation. After a decarboxylation the negative charge is partially absorbed by the PLP (electron sink), leaving an nucleophilic secondary aldimine. The enzymatic reside that favoured the departure of the carboxyl group (say, catalytic lysine) might compete with the SAM though.

In the literature there is no sign of such a reaction: there is a decarboxylative O-methyltransferase (PMID: 22247507) and the various cases of SAM and PLP dependent enzymes, e.g. aminomutases, rely on SAM as a radical donor. There are some enzymes that point towards the possibility of such a C-methyltransferase, such as on an enol-ketone tautomerism (PMID: 5490210 and PMID: 17784761). Nevertheless, such an enzyme, if possible, would require a lot of work and luck to pull off. So the safer option might sound a lot longer, but has a higher chance of working...

Selection

A side question is how to select for better variants. To evolve a strain to make methamphetamine an way to select for one is obviously needed.
The traditional way would be to assay for those compounds by HPLC, but this would mean that the variants would be screened laboriously (especially in light of the optimisation required).
An option for a high-throughput approach is to make a transcription factor that responds only to the substance needed, so that it activates a fluorescent reporter which can be selected by a FACS (eg. PMID: 22276138). Unfortunately, the targets of amphetamines and those of catecholamines are membrane receptors. So the phenylalanine binding TF, tyrR-econded, would be a good candidate for engineering.

Toxicity

The oral murine LD50 of methamphetamine is slightly higher than capsaicin (55 vs. 46 mg/kg), so it is probably non-toxic to bacteria and due to its hydrophobicity it can be phase-separated easily from the aqueous environment. So a least one bit would be straight-forward.

Conclusion

Given the recombination and sequencing for gene identification, the many rounds of engineering and so forth, it would be five years if they are lucky. So metabolic engineering breaking bad would not start even at its fifth season...
And it would be expensive to do and there is no guarantee that their strain would remain safe — copying Walter's formula was an issue, here it would require only a stolen tube.
In brief, it would actually be a real pain to do and take years to cobble together, so unfortunately, metabolic engineering cannot make Walter and Jessie more green and free of precursor woes...