Literature Review

So, if csy1 is not the capsaicin synthase, what do current researchers think is?

The architects of our metabolic map (Mazourek et al, 2009) have written a nice paper describing how they built it (on SolCyc, before it got wrapped up in the rest of MetaCyc). About the synthase, that they leave a fork connected to so tantalizingly, they say,

- CoA-activated fatty acids form 6x more capsaicin in extracts than free CoA and free fatty acids, suggesting that there is a CoA intermediate between the fatty acid and the capsaicin synthase

-AT3, an acyltransferase which is preferentially expressed in the placenta (where the capsaicin accumulates), when knocked out, causes loss-of-pungency, has been a candidate. However, some loss-of-pungency mutants form capsaicinoids when precursors are added exogenously.

-Another point, off the CS topic but of interest to artisan transgeneticists: most of the capsaicinoid pathway has orthologs within tomato (Solanum lycopersicum), which has a great genome sequence and lots of genetic work done. The fruits actually contain vanillin.

Second paper of interest, referred to earlier, is an RNA-sequencing project which contrasts the expression of the pepper pericarp and placenta (Lei, Liu, et al 2012). Their findings?

-61 genes with specific expression in the placenta, many of which (PAL, 4CL, C3H, COMT) have already found a place in the MetaCyc biosynthetic pathway. Only 27 of those 61 genes had homology with some existing gene, indicating that the CS may be among those 34 unknown genes. Homology means that they were able to match the gene to an already-understood family of genes, generally using BLAST or some other search algorithm that matches gene sequences by the way that they line up. Homology is rarely perfect: just because you have something that really looks like an acyltransferase doesn’t mean it will necessarily transfer acyl groups, and even if it does which acyls it will transfer is hard to know.

-Among the 27 with homology, 10 acyltransferase-like genes were found, including AT3. It could be that AT3 does work as a synthase, but it is operating with other acyltransferases with the same activity. They also refer to an acyltransferase gene which Mazourek, et al, identified back in 2005, AY819027, but it is not clear if they found it.  

We can do the same search that these researchers did to find homologous genes. Pubmed has an easy to use BLAST tool, and using it on AY819027 (specifying that we only want to see Capsicum genes), we find a few genes that are very similar (acyltransferases taken from hot peppers around the Pun1 locus), and also a few that are listed as non-pungent varieties.

So regardless of the fact that this is similar to AT3, these genes are clearly not making capsaicin. This does not disprove that they could, however: they could be missense mutants, not be expressed, or else the intermediates before capsaicin may not be being made.

The proof is in the (poblano corn) pudding! If we want to know for sure what gene in our pepper makes it spicy instead of tasting like vanilla, the most definite method we have is to get these genes out of our pepper, express them in some host, and test them in a solution of of vanillylamine and fatty acid. That’s a lot of process steps to understand, so we’ve got some posts cut out for us:

-First we find the 30-some genes listed in RNA profiling paper that might have a synthase activity

-Then we’ll talk about how to purify the DNA from our peppers (which have recently sprouted, and will probably be slightly dissimilar to the genes in the paper)

-Finally, we’ll talk about how we can express these outside of the pepper, separately, so we can test their activity.

It’s a lot more work then we thought we would have to do at the beginning of this paper, but hey, that’s the burden of proof.

Genetics databases, the burden of proof

Hey everybody!

So last post we said we'd go into accessing the genetics of peppers; our goal is to find the sequence of the capsaicin synthase, so we can create some kind of interference in our peppers to prevent it from expressing.

So we can hop over to pubmed and search "capsaicin synthase" under "Nucleotide" and we get seven or eight hits, all 981 bp.

Unfortunately, we've been reading too much recent literature on the subject and this raises red flags: according to an RNA-sequencing profiling project of Lin, Lei, et al, ( 2012)

1: There is no complete sequence of the pepper genome.

2: Within the more limited sequencing projects that have occurred, no one is quite sure what the capsaicin synthase gene is.

Back to the genes we found on pubmed:

They mostly stem from a paper published in PNAS in 2006, which was retracted in 2008.  The paper is fairly exhaustive: they did a column fractionation of placental enzymes, then tested each fraction for capsaicin synthase activity, by putting the enzyme mixture in a solution of vanilllylamine and 8- methyl-nonenoic acid, and testing the amount of capsaicin that resulted on HPLC. 

They then purified the enzymes on a column containing bound vanillylamine, in the hopes that the affinity CS has for vanillylamine would purify it from other enzymes.

Now that they have a fairly pure product, they sequence it, starting with its amino acids, and go to the point of taking their genomic sequence, putting it into an E.coli vector, and making it heterologously. However, although they give a number for the activity of the heterologous csy1, there are no figures. One would think that you would want to blast that piece of proof right up front where everyone can see it. 

The retraction states that the genetic sequence shows homology to a protein kinase found later, by a M. Rapolu. Since the homology is for a small part of this kinase (981 bp for CS of > 3000 bp for the kinase), this suggests that their gene was wrong, although chances are good that they did purify some kind of synthase, before the sequencing part.

This means that there probably isn't any juice to this protein.  It also suggests that they never inserted the gene in E. coli, or didn't get any activity, or something in the media / E. coli spontaneously creates capsaicin, which is why there are no controls. 

But that is somewhat rude. If biotechnology was as easy as taking one beaker and pouring it into another, we wouldn't have to write this blog. Messing with genes is hard. Why is it so hard?

- Most genes are very hard to see, and when you purify them or do PCR on them, they mutate, sometimes in a way that makes them inactive. 

- Although it is easy enough to align genes with other genes that you may have some idea of their function, actually figuring out the function of any particular gene, in the context of the thousands of other genes, can be a combinatorially hard problem.

With that cautionary note, next time we will discuss the candidates for capsaicin synthase, and how we might go about testing them.

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