Throughout human history, microbes have been working for us.
Crops rely on soil bacteria for health and productivity. In Ancient Egypt, bakers and brewers shared yeast cultures as next-door neighbors. Milk sours fast, but with bacteria we convert it to cheese and yoghurt.
Today, we want to expand their repertoire a little bit. What if, instead of fermenting beer, we could ferment cancer drugs?
Synthetic biology harnesses the ease of biological systems in controlling chemicals. One field of synthetic biology is metabolic engineering. Can we engineer a cell’s metabolism to produce medicines, industrial products, or better beer?
What is a Cell?
To an engineer, a cell is a biorefinery, producing hundreds of chemicals from whatever carbon source it had for breakfast. If that cell is a yeast, it produces a lot of ethanol. If it’s an E. coli, the metabolic profile looks different.
Metabolite, chemical, small molecule – for the purposes of this essay, they’re all the same. A cell’s metabolism shuffles carbon from one molecule to another. Glucose is a favorite carbon source. Insert a pathway, and you can get E. coli to eat acetate as well. Add two extra steps, and suddenly you can access hundreds of a particular class of molecules. We know if you grow a cell on acetate instead of glucose, its entire metabolic profile changes. The molecules produced by a cell also depend on its biology – yeast produce ethanol happily, but E. coli find ethanol toxic.
In any case, you feed a cell glucose (or acetate, or cellobiose, or some other carbon source), and the cell converts that into everything it needs to survive. That includes:
- Amino acids
- Quorum sensors
- Chemotactic molecules
- Signaling molecules
- Chemical defenses
This soup of molecules is unique to each cell, and unique to the circumstances (fighting to survive, in equilibrium), its food source (flour, milk, wastewater), and current chemical balance (if there’s a lot of some amino acid sitting around, the cell is not about to waste energy and resources to make more of it.)
With metabolic engineering, we can direct flux into the pathways we like the best.
The flux in yeast, for example, runs from glucose to ethanol. In the engineered E. coli example we looked at above, flux runs from glucose and acetate into terpenoids. Metabolic engineering allows us to redirect flux through favorite pathways and insert new pathways towards molecules novel to the cell.
What is a Pathway?
If cells are our biorefineries, what is the mechanism? For this we recall the central dogma of biology.
All of the shuffling of molecules cells do? Proteins do that. Enzymes do that. We’ve learned how coenzyme A converts pyruvate into acetyl-CoA during glycolysis. We know RuBisCo is the most abundant enzyme on the planet, working diligently to fix carbon dioxide in the Calvin Cycle. These enzymes are not just floating around in the cell; they are actively produced by the cell. They are encoded in the cell’s genes.
That means that we can add new genes to the cell to change which enzymes are made. If we want yeast to make isobutanol instead of ethanol, we’ve got to add a few butanol synthesis genes (and maybe delete the pyruvate decarboxylase, which sends carbon flux into ethanol).
Adding and deleting genes, then, is a tool of the trade for metabolic engineers. Other tricks? It helps to know the cell’s innate biology. You can make isobutanol in yeast or E. coli, but they will require different engineering techniques to optimize production.
What to Feed the Cell?
Let’s shift gears and look at the impact of carbon source. In the review paper Alternative Substrate Metabolism in Yarrowia Lipolytica, scientists outline how the metabolic profile changes in a species of yeast – Y. lipolytica – as it is fed various sugars and oils. From the paper, it appears you can give just about anything with a carbon backbone to Y. lipolytica and it will break it down to use in its metabolism.
Why is this significant? Well, up to now we’ve considered using cells as biorefineries. They can also be used for bioremediation or biofuel purposes.
For example, consider bioremediation of carbon-based compounds. Plastic, oil, phenol – these pollutants all contain carbon that cells can break down and recycle into simple sugars. We can also take carbon-rich sources – say, corn or switchgrass – and engineer cells that consume not only their glucose, but their more difficultly resourced cellulosic carbon products. Y. lipolytica is a favorite candidate to do just that, and because of its oiliness, can convert such tricky carbon compounds into oily biofuels.
Depending on what you feed it, carbon will flow into different metabolic “wells.” Glucose and similar six-carbon sugars (mannose, galactose, fructose) take various paths into glycolysis. While the pathway for the breakdown of the five-carbon sugar xylose is still being pinned down, it and other pentoses go straight to the pentose phosphate pathway (PPP), crucial for nucleotide synthesis. Xylose is a sugar common to potential biofuels like corn, so the ability to break this down for use in basic biology is a coveted one.
The biofuel industry is also interested in lipid-rich waste-products that Y. lipolytica might be able to break down. Lipids aren’t carbohydrates, like the sugars above, but still a rich carbon source that this yeast can use. Olive oil mill wastewater is a by-product of olive oil production, and is a common pollutant in the Mediterranean. Y. lipolytica has been shown to grow on it and so can help with bioremediation (specifically, with breaking down the rich phenolic content). Animal fats are another – very cheap – waste product, but Y. lipolytica can not only grow on them, but naturally produce fairly high titers of citric acids while doing so. Waste cooking oil is another option; Y. lipolytica has already been used to process it for erythritol (usually synthesized from more expensive glucose).
If you’re a biofuels company, all you have to do is find something like this that the yeast can grow on, and engineer it to produce some valuable energy product from these cheap carbon source that would otherwise be thrown away.
This sort of approach is sustainable, protects the environment from discarded pollutants, and protects the environment via by-passing industrial, fossil-fueled methods of synthesizing these chemicals. This is the sort of cellular power that metabolic engineering can tap into.
Spagnuolo, M., Hussain, M. S., Gambill, L., & Blenner, M. (2018). Alternative substrate metabolism in Yarrowia lipolytica. Frontiers in Microbiology, 9(MAY), 1–14. https://doi.org/10.3389/fmicb.2018.01077