The art of blowing up protein

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:

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

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.

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.

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

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.

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.

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.