Isoprenoids are a huge class of molecules – over 50,000 of them – produced naturally by biology. Like many natural compounds, they can be harvested for industrial purposes. Various isoprenoids are used today in medicine, agriculture, flavors, cosmetics, fragrance, and nutrition. In the paper Two-step pathway for isoprenoid synthesis, we ask the question: What if we could use biology to synthesize them?
This paper is also a study in metabolic engineering, an application within synthetic biology. A cell’s metabolism involves lots of compounds – such as isoprenoids – and some of them are useful for human consumption. The general strategy is: (1) add carbon (usually glucose), (2) direct the flux towards some product, and (3) boast your cell’s high titer. As such, we can here learn a little about the tests, strategies, and problem solving that goes into building industries with biology instead of petrochemistry. Why is that such a powerful idea? Petrochemistry means making products from fossil fuels, and much of modern industry is built directly upon it.
First things first. As a metabolic engineering paper, there are lots of words in here describing steps of the metabolic pathways, and I loathe them. I always do. Because everyone knows that a diagram makes so much more sense than talking about a bunch of enzymes and molecules and reactions.
Now, I am going to be the jerk here and also talk about the metabolic pathways with my words, instead of showing you pictures. I will do this for the simple reason that this website is a hobby and I can’t throw $30 at copyright access every time I want to show you all a picture. However, if you wish to understand anything in this paper or even in my summary of it here, you will consider taking a look at the two-step pathway to isoprenoids shown in Fig. 1A.
So what exactly is the problem the researchers here are trying to solve? Isoprenoids are this treasure trove of industrially relevant materials. Early on, they build a pathway towards isoprenoids in general, but later in the paper, they make specific isoprenoids like lycopene (the red carotenoid in tomatoes) and taxadiene (the precursor for taxol, a chemotherapeutic drug). So these are great molecules to have around, and we want to make them efficiently with microbes (in this paper, we’re using E. coli). The native isoprenoid pathway in E. coli is the MEP pathway.
The MEP is definitely something worth knowing about if you want to understand a cell’s metabolism. But for the purposes of this introduction, let’s just call it one of the many wonderful pathways inside the cell, like photosynthesis or the Krebs cycle or Amino Acid synthesis or the PPP sending flux straight into nucleotide synthesis. The MEP is just another one of those internal pathways, used to produce this class of biomolecules called isoprenoids.
To a metabolic engineer, the cell is like a fancy plumbing system, with giant pathways of pipes flowing into all sorts of different sinks and exported products. You input carbon, and it flows through different pathways – glycolysis, amino acid synthesis, DNA synthesis, isoprenoid synthesis, etc. The engineer wants to fiddle with the valves, cut out unnecessary piping, and direct the flux to the product of interest. But this must be done without damaging the cell’s core metabolism too much (if you break a pipe on the Krebs cycle, for example, you’ll end up killing the cell).
So the MEP is the native isoprenoid pathway. But unfortunately, it has some inefficiencies. Enzymes in this pathway contain iron, making them sensitive to oxygen. Oxygen exposure inactivates the enzyme. When this happens, carbon flowing towards others molecules, soon exported out of the cell. In other words, inefficient enzymes lead to carbon loss (we’ve seen strategies to deal with carbon loss before.)
So with MEP, you feed the cell carbon, but as enzymes start breaking down, carbon is stolen away from the isoprenoid pathway it’s supposed to be fueling.
To solve this, the researchers engineered E. coli with a new, more efficient, two-step pathway. And to test it, they used it to produce actual isoprenoids like lycopene, taxadiene, limonene, and amorphadiene. The end result? A new-and-improved bio-based method for producing isoprenoids.
So what is this fancy new streamlined pathway?
Well, remember, we must first consult the actual diagram.
Isoprenoids need two inputs: molecules called IPP and DMAPP.
To get there, you can start by adding isoprenol and prenol (cheap carbon substrates, which the researchers use instead of glucose). From there, you only need two steps: isoprenol and prenol are converted to IP and DMAP, and then IP and DMAP are converted to IPP and DMAPP.
First, a kinase is needed. A kinase is an enzyme that will add a phosphate group to its substrate. They actually tested a bunch of kinases in case some were really slow and others faster; you get a list of all the enzymes tested in Fig. 1B. Sure enough, many of these performed poorly, and only one did a stellar job at catalyzing this chemical reaction. In Fig. 1C, ScCK (the yeast cell S. cerevisiae‘s choline kinase) is identified as winning the race for the first step.
Now for the second step. DMAP gets a second phosphate group to become DMAPP, and IP gets the same to become IPP. In this case, however, the researchers went for a more specific kinase, the IPK enzyme from the plant A. thaliana, which is already known to be a great team player for this particular reaction.
As an extra detail, the more specific IPK stands in contrast to cerevisiae‘s choline kinase, which is described in Fig. 1A as a “promiscuous kinase.” We use the word “promiscuous” for enzymes that will react with just about anything. IPK is picky, preferring to interact only with select molecules as its substrate, but this choline kinase will give away phosphate groups to any substrate that will take them.
So IPK does the trick for step two, and we have the DMAPP and IPP needed to synthesize isoprenoids. You may also notice the ATP’s showing up at most of the chemical reactions; ATP is, of course, adenosine triphosphate, so it’s got phosphate groups to spare. The kinases work by stealing a phosphate from ATP and putting it on their substrates, leaving an ADP (diphosphate) behind.
The researchers have one more trick up their sleeve. To really make sure this pathway is efficient, you want to make sure the reaction is balanced. So to finish it off, they added one more enzyme: IDI, a non-directional enzyme that evens out DMAPP and IPP, Le Chatelier style.
They named their new pathway the IUP (isopentenol utilization pathway), and then they had to test it.
Chatzivasileiou, A. O., Ward, V., Edgar, S. M. B., & Stephanopoulos, G. (2019). Two-step pathway for isoprenoid synthesis. Proceedings of the National Academy of Sciences of the United States of America, 116(2), 506–511. https://doi.org/10.1073/pnas.1812935116